Antibodies for inhibiting blood coagulation and methods of use thereof

ABSTRACT

The invention includes antibodies that provide superior anti-coagulant activity by binding native human TF with high affinity and specificity. Antibodies of the invention can effectively inhibit blood coagulation in vivo. Antibodies of the invention can bind native human TF, either alone or present in a TF:FVIIa complex, effectively preventing factor X or FIX binding to TF or that complex, and thereby reducing blood coagulation. Preferred antibodies of the invention specifically bind a conformational epitope predominant to native human TF, which epitope provides an unexpectedly strong antibody binding site. Also provided are humanized antibodies and fragments thereof that bind to the TF.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 09/990,586, filed on Nov. 21, 2001, which claims priority toU.S. Provisional Application No. 60/343,306, filed on Oct. 29, 2001,which application is related to U.S. patent application Ser. No.09/293,854 filed on Apr. 16, 1999 (now U.S. Pat. No. 6,555,319), whichapplication is a divisional of U.S. Ser. No. 08/814,806 (now U.S. Pat.No. 5,986,065). The disclosures of said U.S. patent application Ser. No.09/990,586 as filed on Nov. 21, 2001, U.S. Provisional Application No.60/343,306 as filed on Oct. 29, 2001, U.S. Pat. No. 6,555,319, and U.S.Pat. No.5,986,065 are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel human tissue factor antibodiesand methods of using the antibodies to inhibit tissue factor-relatedfunctions such as blood coagulation, angiogenesis, tumor metastasis, andinflammation. In particular, the invention relates to novel antibodiesthat can specifically bind native human tissue factor with high affinityand prevent factor X or factor IX binding and activation. The antibodiesof the invention are useful for a variety of applications, particularlyfor reducing blood coagulation in vivo.

2. Background

Blood clotting assists homeostasis by minimizing blood loss. Generally,blood clotting requires vessel damage, platelet aggregation, activationof coagulation factors and inhibition of fibrinolysis. The coagulationfactors act through a cascade that relates the vessel damage toformation of a blood clot (see generally L. Stryer, Biochemistry, 3rdEd, W.H. Freeman Co., New York; and A. G. Gilman et al., ThePharmacological Basis of Therapeutics, 8th Edition, McGraw Hill Inc.,New York, pp. 1311-1331).

There is general agreement that factor X (FX) activation to factor Xa(FXa) (or factor IX activation to factor IXa) is a critical step in theblood coagulation process. Generally, FX (or FIX) is converted to FXa(or FIXa) by binding a catalytically active complex that includes“tissue factor” (TF). TF is a controllably-expressed cell membraneprotein that binds factor VII/VIIa to produce the catalytically activecomplex (TF:FVIIa). A blood clot follows FXa-mediated (or FIXa)activation of prothrombin. Blood clotting can be minimized byinactivation of TF to non-native forms which cannot optimally producethe TF:FVIIa complex. Excessive activation of the coagulation cascadethrough formation of FXa (or FIXa) is believed to contribute to variousthromboses including restenosis.

Thrombosis may be associated with invasive medical procedures such ascardiac surgery (e.g. angioplasty), abdominothoracic surgery, arterialsurgery, peripheral vascular bypass grafts, deployment of animplementation (e.g., a stent or catheter), or endarterectomy. Further,thrombosis may accompany various thromboembolic disorders andcoagulopathies such as stroke, pulmonary embolism (e.g., atrialfibrillation with embolization), coronary artery disease or acutecoronary syndromes (e.g., unstable angina or myocardial infarction),atherosclerosis or other thrombo-occlusive disorders, deep veinthrombosis and disseminated intravascular coagulation, respectively.Manipulation of body fluids can also result in an undesirable thrombus,particularly in blood transfusions or fluid sampling, as well asprocedures involving extracorporeal circulation (e.g., cardiopulmonarybypass surgery) and renal dialysis.

Anti-coagulants are frequently used to alleviate or avoid blood clotsassociated with thrombosis. Blood clotting often can be minimized oreliminated by administering a suitable anti-coagulant or mixturethereof, including one or more of a coumarin derivative (e.g., warfarin,Coumadin or dicumarol) or a charged polymer (e.g., heparin, lowmolecular weight heparin, hirudin or hirulog) or anti-platelet agents(e.g., ReoPro, Integrilin, Aggrestat, Plavix, Ticlid or aspirin). Seee.g., Gilman et al., supra, R. J. Beigering et al., Ann. Hematol.,72:177 (1996); J. D. Willerson, Circulation, 94:866 (1996).

However, use of anti-coagulants is often associated with side effectssuch as hemorrhaging, re-occlusion, “white-clot” syndrome, irritation,birth defects, thrombocytopenia and hepatic dysfunction. Long-termadministration of anti-coagulants can particularly increase risk oflife-threatening illness (see e.g., Gilman et al., supra).

Certain antibodies with anti-platelet activity have also been used toalleviate various thromboses. For example, ReoPro®™ is a therapeuticantibody fragment that is routinely administered to alleviate variousthromboembolic disorders such as those arising from angioplasty,myocardial infarction, unstable angina and coronary artery stenoses.Additionally, ReoPro® can be used as a prophylactic to reduce the riskof myocardial infarction and angina (J. T. Willerson, Circulation,94:866 (1996); M. L. Simmons et al., Circulation, 89:596 (1994)).

Certain anti-coagulant antibodies are also known. Particularly, certainTF-binding antibodies have been reported to inhibit blood coagulation,presumably by interfering with assembly of a catalytically activeTF:FVIIa complex (see e.g., Jeske et al., SEM in THROM. and HEMO, 22:213(1996); Ragni et al., Circulation, 93:1913 (1996); European Patent No. 0420 937 B1; W. Ruf et al., Throm. Haemosp., 66:529 (1991); M. M. Fiorieet al., Blood, 8:3127 (1992)).

However, current TF-binding antibodies exhibit significant disadvantageswhich can minimize their suitably as anti-coagulants. For example,current TF-binding antibodies do not exhibit sufficient binding affinityfor optimal anti-coagulant activity. Accordingly, for many thromboticconditions, to compensate for such ineffective binding affinities,unacceptably high antibody levels must be administered to minimize bloodcoagulation.

It would thus be desirable to have an anti-coagulant antibody that bindsnative human TF with high affinity and selectivity to thereby inhibitundesired blood coagulation and the formation of blood clots. It wouldbe further desirable to have such an anti-coagulant antibody thatprevents the binding of factor X (or factor IX) to TF:FVIIa complex.

SUMMARY OF THE INVENTION

We have now discovered antibodies that provide superior anti-coagulantactivity by binding native human TF with high affinity and specificity.Antibodies of the invention can effectively inhibit blood coagulation invivo. Antibodies of the invention can bind native human TF, either aloneor present in a TF:FVIIa complex, effectively preventing factor X (orfactor IX) binding to TF or that complex, and thereby reducing bloodcoagulation.

Preferred antibodies of the invention are monoclonal and specificallybind a conformational epitope predominant to native human TF, whichepitope provides a site for the unexpectedly strong antibody binding.Indeed, preferred antibodies of the invention bind to native human TF atleast about 5 times greater, more typically at least about ten timesgreater than the binding affinity exhibited by prior anti-coagulantantibodies. Additionally, preferred antibodies of the invention areselective for native human TF, and do not substantially bind non-nativeor denatured TF. H36.D2.B7 (secreted by hybridoma ATCC HB-12255 andoften referred to as H36) is an especially preferred antibody of theinvention.

Preferred antibodies of the invention bind TF so that FX (or FIX) doesnot effectively bind to the TF:FVIIa complex whereby FX (or FIX) is noteffectively converted to its activated form (FXa or FIXa). Preferredantibodies of the invention can inhibit TF function by effectivelyblocking FX (or FIX) binding or access to TF molecules. See, forinstance, the results of Example 3 which follows.

Preferred antibodies of the invention also do not significantly inhibitthe interaction or binding between TF and factor VIIa, or inhibitactivity of a TF:FVIIa complex with respect to materials other than FXand Factor IX. See, for instance, the results of Example 4 whichfollows.

The invention also provides nucleic acids that encode antibodies of theinvention. Nucleic acid and amino acid sequences (SEQ ID NOS: 1-4) ofvariable regions of H36.D2.B7 are set forth in FIGS. 1A and 1B of thedrawings.

In preferred aspects, the invention provides methods for inhibitingblood coagulation and blood clot formation, and methods for reducinghuman TF levels.

In general, antibodies of the invention will be useful to modulatevirtually any biological response mediated by FX (or FIX) binding to TFor the TF:FVIIa complex, including blood coagulation as discussed above,inflammation, tumor angiogenesis and metastasis, and other disorders.

Antibodies of the invention are particularly useful to alleviate variousthromboses, particularly to prevent or inhibit restenosis, or otherthromboses following an invasive medical procedure such as arterial orcardiac surgery (e.g., angioplasty). Antibodies of the invention alsocan be employed to reduce or even effectively eliminate thromboticocclusion arising from activation of blood coagulation in suchnon-surgical cardiovascular conditions including but not limited tocoronary artery disease, acute coronary syndromes (e.g., unstable anginaand myocardial infarction) and atherosclerosis. Antibodies of theinvention also can be employed to reduce or even effectively eliminateblood coagulation arising from use of medical implementation (e.g., acatheter, stent or other medical device). Preferred antibodies of theinvention will be compatible with many anti-coagulant, anti-platelet andthrombolytic compositions, thereby allowing administration in a cocktailformat to boost or prolong inhibition of blood coagulation.

Antibodies of the invention also can be employed as an anti-coagulant inextracorporeal circulation of a mammal, particularly a human subject. Insuch methods, one or more antibodies of the invention is administered tothe mammal in an amount sufficient to inhibit blood coagulation prior toor during extracorporeal circulation such as may be occur withcardiopulmonary bypass surgery, organ transplant surgery or otherprolonged surgeries.

Antibodies of the invention also can be used as a carrier for drugs,particularly pharmaceuticals targeted for interaction with a blood clotsuch as strepokinase, tissue plasminogen activator (t-PA) or urokinase.Similarly, antibodies of the invention can be used as a cytotoxic agentby conjugating a suitable toxin to the antibody. Conjugates ofantibodies of the invention also can be used to reduce tissue factorlevels in a mammal, particularly a human, by administering to the mammalan effective amount of an antibody of the invention which is covalentlylinked to a cytotoxic agent or an effector molecule to providecomplement-fixing ability and antibody-dependent cell-mediatedcytotoxicity, whereby the antibody conjugate contacts cells expressingtissue factor to thereby reduce tissue factor levels in the mammal.

Antibodies of the invention also can be employed in in vivo diagnosticmethods including in vivo diagnostic imaging of native human TF.

Antibodies of the invention also can be used in in vitro assays todetect native TF in a biological sample including a body fluid (e.g.,plasma or serum) or tissue (e.g., a biopsy sample). More particularly,various heterogeneous and homogeneous immunoassays can be employed in acompetitive or non-competitive format to detect the presence andpreferably an amount of native TF in the biological sample.

Such assays of the invention are highly useful to determine the presenceor likelihood of a patient having a blood coagulation or a blood clot.That is, blood coagulation is usually accompanied by and the result ofTF expression on cell surfaces such as monocytes, macrophages, andendothelial cells lining the vasculature. Thus, the detection of TF in abody fluid sample by an assay of the invention will be indicative ofblood coagulation.

Antibodies of the invention also can be used to prepare substantiallypure native TF, particularly native human TF, from a biological sample.Antibodies of the invention also can be used for detecting and purifyingcells which express native TF.

Antibodies of the invention also can be employed as a component of adiagnostic kit, e.g. for detecting and preferably quantitating native TFin a biological sample.

The invention also provides humanized antibodies that bind specificallyto human tissue factor (TF) to form a complex. In a preferredembodiment, blood factor X or factor IX binding to the complex issignificantly inhibited. Preferably, the humanized antibody includes atleast one murine complementarity determining region (CDR), preferablyone, two, three or four of such murine CDRs. Further provided are TFbinding fragments of such humanized antibodies.

In another aspect, the invention provides methods of inhibiting bloodcoagulation in a mammal that include administering to the mammal aneffective amount of the humanized antibody or fragment thereof thatbinds specifically to human tissue factor (TF) to form a complex. Apreferred antibody for use in the method significantly reduces factor Xor factor IX binding to the complex. Preferred methods further includeforming a specific complex between the antibody and the TF or TF:FVIIacomplex to inhibit the blood coagulation.

The invention also provides methods of inhibiting blood coagulation in amammal that include administering to the mammal, an effective amount ofa humanized antibody or fragment thereof comprising at least one murinecomplementarity determining region (CDR). A preferred humanized antibodyfor use with the method binds specifically to human tissue factor (TF)to form a complex. Preferably, factor X or factor IX binding to thecomplex is significantly reduced. Preferred methods further includeforming a specific complex between the antibody and the TF to inhibitthe blood coagulation.

Other aspects of the invention are discussed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows the nucleic acid (SEQ ID NOS: 1 and 3) and aminoacid (SEQ ID NOS:2 and 4) sequences of light chain and heavy chainvariable regions of H36.D2.B7 with hypervariable regions (CDRs orComplementarity Determining Regions) underlined (single underline fornucleic acid sequences and double underline for amino acid sequences)(SEQ ID NOS: 5-10, respectively, in order of appearance).

FIG. 2 shows association (K_(a)) and disassociation (K_(d)) constants ofanti-tissue factor antibodies as determined by ELISA or BIACoreanalysis.

FIG. 3 shows inhibition of TF:FVIIa complex mediated FX activation bypre-incubation with anti-tissue factor antibodies.

FIG. 4 shows inhibition of TF:FVIIa activity toward the FVIIa-specificchromogenic substrate S-2288 by anti-tissue factor antibodies.

FIG. 5 shows the capacity of the H36 antibody to increase prothrombintime (PT) in a TF-initiated coagulation assay.

FIGS. 6A and 6B graphically show the relationship between FXa formationand molar ratio of the H36 antibody and rHTF. FIG. 6A: H36 waspre-incubated with the TF:FVIIa complex prior to adding FX. FIG. 6B:H36, TF:FVIIa and FX were added simultaneously.

FIG. 7 shows inhibition of TF:FVIIa activity by the H36 antibody in aJ-82 cell activation assay.

FIGS. 8A and 8B are representations of dot blots showing that the H36antibody binds a conformational epitope on rhTF. Lane 1-native rHTF,Lane 2-native rhTF treated with 8M urea, Lane 3-native rHTF treated with8M urea and 5 mM DTT. In FIG. 8A, the blot was exposed for approximately40 seconds, whereas in FIG. 8B, the blot was exposed for 120 seconds.

FIGS. 9A-B are drawings showing human IgG1-cH36 HC variable regioncloning and expression vectors. HC cloning vector (9A) and HC expressionvector (9B).

FIGS. 9C-D are drawings showing human IgG4-cH36 HC variable regioncloning and expression vectors. HC cloning vector (9C) and HC expressionvector (9D).

FIGS. 10A-B are drawings showing cH36 LC variable region cloning andexpression vectors. LC cloning vector (10A) and LC expression vector(10B).

FIG. 11 is a drawing showing a plasmid map of humanized anti-TF IgG1antibody expression vector (pSUN 34).

FIGS. 12A-D are drawings showing sequences of partially and fullyhumanized light chain (LC) variable regions. FIG. 12 sequencescorrespond to SEQ ID NOS: 72-82, respectively, in order of appearance.Light chain CDR sequences CDR sequences of cH36 are shown in FIGS. 12B-D(fragment of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7, respectively).Sequence named “LC-09” (SEQ ID NO: 79) is representative of a fullyhumanized LC framework region.

FIGS. 13A-D are sequences of partially and fully humanized heavy chain(LC) variable regions. FIG. 13A sequences correspond to SEQ ID NOS:83-96, respectively, in order of appearance. Heavy chain CDR sequencesfor cH36 and HC-08 are shown in FIGS. 13B-D (SEQ ID NO: 8, SEQ ID NOS: 9and 101, and SEQ ID NO: 10, respectively, in order of appearance).Sequence named “HC-08” (SEQ ID NO: 91) is fully humanized HC frameworkregion.

FIGS. 14A-B (SEQ ID NOS: 97 and 98, respectively, in order ofappearance) are drawings showing humanized IgG one anti-tissue factorantibody (hOAT (IgG1) constant regions.

FIGS. 15A-B (SEQ ID NOS: 99-100, respectively, in order of appearance)are drawings showing humanized IgG four anti-tissue factor antibody(hFAT) (IgG4) constant regions.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, preferred antibodies of the invention exhibitsubstantial affinity for native human TF. In particular, preferredantibodies of the invention exhibit an association constant (K_(a), M⁻¹)for native human TF of at least about 1×10⁸ as determined by surfaceplasmon analysis (particularly, BIACore analysis in accordance with theprocedures of Example 1 which follows), more preferably at least about5×10⁸ as determined by surface plasmon analysis, still more preferably aK_(a) (K_(a), M⁻¹) for native human TF of at least about 1×10¹⁰ asdetermined by surface plasmon resonance analysis. Such substantialbinding affinity of antibodies of the invention contrast sharply frommuch lower binding affinities of previously reported antibodies.

In this regard, a quite low of effective concentration of an antibody ofthe invention can be employed, e.g. a relatively low concentration ofantibody can be employed to inhibit TF function as desired (e.g. atleast about 95, 98 or 99 percent inhibition) in an in vitro assay suchas described in Example 3 which follows.

The preferred antibodies are also highly specific for native human TF,and preferably do not substantially bind with non-native TF. Preferredantibodies do not substantially bind non-native TF or otherimmunologically unrelated molecules as determined, e.g. by standard dotblot assay (e.g. no or essentially no binding to non-native TF visuallydetected by such dot blot assay). References herein to “non-native TF”mean a naturally-occurring or recombinant human TF that has been treatedwith a chaotropic agent so that the TF is denatured. Typical chaotropicagents include a detergent (e.g. SDS), urea combined with dithiothreotolor P-mercaptoethanol; guanidine hydrochloride and the like. The H36,H36.D2 or H36.D2.B7 antibody does not substantially bind to suchnon-native TF. See, for instance, the results of Example 8 which followsand is a dot blot assay.

As discussed above, preferred antibodies of the invention also bind withTF so that FX (or FIX) does not effectively bind to the TF:FVIIa complexwhereby FX (or FIX) is not effectively converted to its activated form(FXa or FIXa). Particularly preferred antibodies of the invention willstrongly inhibit FX activation by a TF:FVIIa complex, e.g. an inhibitionof at least about 50%, more preferably at least about 80%, and even morepreferably at least about 90% or 95%, even at low TF concentrations suchas less than about 1.0 nM TF, or even less than about 0.20 nM or 0.10 nMTF, as determined by a standard in vitro binding assay such as that ofExample 3 which follows and includes contacting FX (or FIX) with a TF:FVIIa complex both in the presence (i.e. experimental sample) andabsence (i.e. control sample) of an antibody of the invention anddetermining the percent difference of conversion of FX to FXa (or FIX toFIXa) between the experimental and control samples.

Antibodies of the invention are preferably substantially pure when usedin the disclosed methods and assays. References to an antibody being“substantially pure” mean an antibody or protein which has beenseparated from components which naturally accompany it. For example, byusing standard immunoaffinity or protein A affinity purificationtechniques, an antibody of the invention can be purified from ahybridoma culture by using native TF as an antigen or protein A resin.Similarly, native TF can be obtained in substantially pure form by usingan antibody of the invention with standard immunoaffinity purificationtechniques. Particularly, an antibody or protein is substantially purewhen at least 50% of the total protein (weight % of total protein in agiven sample) is an antibody or protein of the invention. Preferably theantibody or protein is at least 60 weight % of the total protein, morepreferably at least 75 weight %, even more preferably at least 90 weight%, and most preferably at least 98 weight % of the total material.Purity can be readily assayed by known methods such as SDS (PAGE) gelelectrophoresis, column chromatography (e.g., affinity chromatography)or HPLC analysis.

The nucleic acid (SEQ ID NOS: 1 and 3) and amino acid (SEQ ID NOS: 2 and4) sequences of a preferred antibody of the invention (H36.D2.B7) areshown in FIGS. 1A and 1B of the drawings. SEQ ID NOS: 1 and 2 are thenucleic acid and amino acid respectively of the light chain variableregion, and SEQ ID NOS: 3 and 4 are the nucleic acid and amino acidrespectively of the heavy chain variable region, with hypervariableregions (CDRs or Complementarity Determining Regions) underlined in allof those sequences.

Additional preferred antibodies of the invention will have substantialamino acid sequence identity to either one or both of the light chain orheavy sequences shown in FIGS. 1A and 1B. More particularly, preferredantibodies include those that have at least about 70 percent homology(amino acid sequence identity) to SEQ ID NOS: 2 and/or 4, morepreferably about 80 percent or more homology to SEQ ID NOS: 2 and/or 4,still more preferably about 85, 90 or 95 percent or more homology to SEQID NOS: 2 and/or 4.

Preferred antibodies of the invention will have high amino acid sequenceidentity to hypervariable regions (shown with double underlining inFIGS. 1A and 1B) of SEQ ID NOS: 2 and 4). Especially preferredantibodies of the invention will have one, two or three hypervariableregions of a light chain variable region that have high sequenceidentity (at least 90% or 95% amino acid sequence identity) to or be thesame as one, two or three of the corresponding hypervariable regions ofthe light chain variable region of H36.D2.B7 (those hypervariableregions shown with underlining in FIG. 1A and are the following: 1)LASQTID (SEQ ID NO: 5); 2) AATNLAD (SEQ ID NO: 6); and 3) QQVYSSPFT (SEQID NO: 7)).

Especially preferred antibodies of the invention also will have one, twoor three hypervariable regions of a heavy chain variable region thathave high sequence identity (at least 90% or 95% amino acid sequenceidentity) to or be the same as one, two or three of the correspondinghypervariable regions of the heavy chain variable region of H36.D2.B7(those hypervariable regions shown with underlining in FIG. 1B and arethe following: 1) TDYNVY (SEQ ID NO: 8); 2) YIDPYNGITIYDQNFKG (SEQ IDNO: 9); and 3) DVTTALDF (SEQ ID NO: 10).

Nucleic acids of the invention preferably are of a length sufficient(preferably at least about 100, 200 or 250 base pairs) to bind to thesequence of SEQ ID NO: 1 and/or SEQ ID NO: 3 under the followingmoderately stringent conditions (referred to herein as “normalstringency” conditions): use of a hybridization buffer comprising 20%formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at atemperature of 37° C. and remaining bound when subject to washing oncewith that SSC buffer at 37° C.

More preferably, nucleic acids of the invention (preferably at leastabout 100, 200 or 250 base pairs) will bind to the sequence of SEQ IDNO: 1 and/or SEQ ID NO: 3 under the following highly stringentconditions (referred to herein as “high stringency” conditions): use ofa hybridization buffer comprising 20% formamide in 0.9M saline/0.09Msodium citrate (SSC) buffer at a temperature of 42° C. and remainingbound when subject to washing twice with that SSC buffer at 42° C.

Nucleic acids of the invention preferably comprise at least 20 basepairs, more preferably at least about 50 base pairs, and still morepreferably a nucleic acid of the invention comprises at least about 100,200, 250 or 300 base pairs.

Generally preferred nucleic acids of the invention will express anantibody of the invention that exhibits the preferred binding affinitiesand other properties as disclosed herein.

Preferred nucleic acids of the invention also will have substantialsequence identity to either one or both of the light chain or heavysequences shown in FIGS. 1A and 1B. More particularly, preferred nucleicacids will comprise a sequence that has at least about 70 percenthomology (nucleotide sequence identity) to SEQ ID NOS: 1 and/or 3, morepreferably about 80 percent or more homology to SEQ ID NOS: 1 and/or 3,still more preferably about 85, 90 or 95 percent or more homology to SEQID NOS: 1 and/or 3.

Particularly preferred nucleic acid sequences of the invention will havehigh sequence identity to hypervariable regions (shown with underliningin FIGS. 1A and 1B) of SEQ ID NOS: 1 and 3). Especially preferrednucleic acids include those that code for an antibody light chainvariable region and have one, two or three sequences that code forhypervariable regions and have high sequence identity (at least 90% or95% nucleotide sequence identity) to or be the same as one, two or threeof the sequences coding for corresponding hypervariable regions ofH36.D2.B7 (those hypervariable regions shown with underlining in FIG. 1Aand are the following: 1) CTGGCAAGTCAGACCATTGAT (SEQ ID NO: 11); 2)GCTGCCACC AACTTGGCAGAT (SEQ ID NO: 12); and 3) CAACAAGTTTACAGTTCTCCATTCACGT (SEQ ID NO: 13)).

Especially preferred nucleic acids also code for an antibody heavy chainvariable region and have one, two or three sequences that code forhypervariable regions and have high sequence identity (at least 90% or95% sequence identity) to or be the same as one, two or three of thesequences coding for corresponding hypervariable regions of H36.D2.B7(those hypervariable regions shown with underlining in FIG. 1B and arethe following: 1) ACTGACTACAACGTGTAC (SEQ ID NO: 14); 2) TATATTGATCCTTACAATGGTATTACTATCTACGACCAGAACTTCAAGGGC (SEQ ID NO: 15); and 3)GATGTGACTACGGCCCTTGACTTC (SEQ ID NO: 16)).

Nucleic acids of the invention are isolated, usually constitutes atleast about 0.5%, preferably at least about 2%, and more preferably atleast about 5% by weight of total nucleic acid present in a givenfraction. A partially pure nucleic acid constitutes at least about 10%,preferably at least about 30%, and more preferably at least about 60% byweight of total nucleic acid present in a given fraction. A pure nucleicacid constitutes at least about 80%, preferably at least about 90%, andmore preferably at least about 95% by weight of total nucleic acidpresent in a given fraction.

Antibodies of the invention can be prepared by techniques generallyknown in the art, and are typically generated to a purified sample ofnative TF, typically native human TF, preferably purified recombinanthuman tissue factor (rhTF). Truncated recombinant human tissue factor or“rhTF” (composed of 243 amino acids and lacking the cytoplasmic domain)is particularly preferred to generate antibodies of the invention. Theantibodies also can be generated from an immunogenic peptide thatcomprises one or more epitopes of native TF that are not exhibited bynon-native TF. References herein to “native TF” include such TF samples,including such rhTF. As discussed above, monoclonal antibodies aregenerally preferred, although polyclonal antibodies also can beemployed.

More particularly, antibodies can be prepared by immunizing a mammalwith a purified sample of native human TF, or an immunogenic peptide asdiscussed above, alone or complexed with a carrier. Suitable mammalsinclude typical laboratory animals such as sheep, goats, rabbits, guineapigs, rats and mice. Rats and mice, especially mice, are preferred forobtaining monoclonal antibodies. The antigen can be administered to themammal by any of a number of suitable routes such as subcutaneous,intraperitoneal, intravenous, intramuscular or intracutaneous injection.The optimal immunizing interval, immunizing dose, etc. can vary withinrelatively wide ranges and can be determined empirically based on thisdisclosure. Typical procedures involve injection of the antigen severaltimes over a number of months. Antibodies are collected from serum ofthe immunized animal by standard techniques and screened to findantibodies specific for native human TF. Monoclonal antibodies can beproduced in cells which produce antibodies and those cells used togenerate monoclonal antibodies by using standard fusion techniques forforming hybridoma cells. See G. Kohler, et al., Nature, 256:456 (1975).Typically this involves fusing an antibody-producing cell with animmortal cell line such as a myeloma cell to produce the hybrid cell.Alternatively, monoclonal antibodies can be produced from cells by themethod of Huse, et al., Science, 256:1275 (1989).

One suitable protocol provides for intraperitoneal immunization of amouse with a composition comprising purified rhTF complex conducted overa period of about two to seven months. Spleen cells then can be removedfrom the immunized mouse. Serum from the immunized mouse is assayed fortiters of antibodies specific for rhTF prior to excision of spleencells. The excised mouse spleen cells are then fused to an appropriatehomogenic or heterogenic (preferably homogenic) lymphoid cell linehaving a marker such as hypoxanthine-guanine phosphoribosyltransferasedeficiency (HGPRT⁻) or thymidine kinase deficiency (TK⁻). Preferably amyeloma cell is employed as the lymphoid cell line. Myeloma cells andspleen cells are mixed together, e.g. at a ratio of about 1 to 4 myelomacells to spleen cells. The cells can be fused by the polyethylene glycol(PEG) method. See G. Kohler, et al., Nature, supra. The thus clonedhybridoma is grown in a culture medium, e.g. RPMI-1640. See G. E. More,et al., Journal of American Medical Association, 199:549 (1967).Hybridomas, grown after the fusion procedure, are screened such as byradioimmunoassay or enzyme immunoassay for secretion of antibodies thatbind specifically to the purified rhTF, e.g. antibodies are selectedthat bind to the purified rhTF, but not to non-native TF. Preferably anELISA is employed for the screen. Hybridomas that show positive resultsupon such screening can be expanded and cloned by limiting dilutionmethod. Further screens are preferably performed to select antibodiesthat can bind to rhTF in solution as well as in a human fluid sample.The isolated antibodies can be further purified by any suitableimmunological technique including affinity chromatography. A hybridomaculture producing the particular preferred H36.D2.B7 antibody has beendeposited pursuant to the Budapest Treaty with the American Type CultureCollection (ATCC) at 12301 Parklawn Drive, Rockville, Md., 10852. Thehybridoma culture was deposited with the ATCC on Jan. 8, 1997 and wasassigned Accession Number ATCC HB-12255.

For human therapeutic applications, it may be desirable to producechimeric antibody derivatives, e.g. antibody molecules that combine anon-human animal variable region and a human constant region, to therebyrender the antibodies less immunogenic in a human subject than thecorresponding non-chimeric antibody. A variety of types of such chimericantibodies can be prepared, including e.g. by producing human variableregion chimeras, in which parts of the variable regions, especiallyconserved regions of the antigen-binding domain, are of human origin andonly the hypervariable regions are of non-human origin. See alsodiscussions of humanized chimeric antibodies and methods of producingsame in S. L. Morrison, Science, 229:1202-1207 (1985); Oi et al.,BioTechniques, 4:214 (1986); Teng et al., Proc. Natl. Acad. Sci. U.S.A.,80:7308-7312 (1983); Kozbor et al., Immunology Today, 4:7279 (9183);Olsson et al., Meth. Enzymol., 9:3-16 (1982). Additionally, transgenicmice can be employed. For example, transgenic mice carrying humanantibody repertoires have been created which can be immunized withnative human TF. Splenocytes from such immunized transgenic mice canthen be used to create hybridomas that secrete human monoclonalantibodies that specifically react with native human TF as describedabove. See N. Lonberg et al., Nature, 368:856-859 (1994); L. L. Green etal., Nature Genet., 7:13-21 (1994); S. L. Morrison, Proc. Natl. Acad.Sci. U.S.A., 81:6851-6855 (1994).

Nucleic acids which code for the antibodies-of the invention also can beprepared by polymerase chain reaction (see primers disclosed in Example1 which follows). See generally, Sambrook et al., Molecular Cloning (2ded. 1989). Such nucleic acids also can be synthesized by known methods,e.g. the phosphate triester method (see Oligonucleotide Synthesis, IRLPress (M. J. Gait, ed., 1984)), or by using a commercially availableautomated oligonucleotide synthesizer. Such a prepared nucleic acid ofthe invention can be employed to express an antibody of the invention byknown techniques. For example, a nucleic acid coding for an antibody ofthe invention can be incorporated into a suitable vector by knownmethods such as by use of restriction enzymes to make cuts in the vectorfor insertion of the construct followed by ligation. The vectorcontaining the inserted nucleic acid sequence, suitably operably linkedto a promoter sequence, is then introduced into host cells forexpression. See, generally, Sambrook et al., supra. Selection ofsuitable vectors can be made empirically based on factors relating tothe cloning protocol. For example, the vector should be compatible with,and have the proper replicon for the host cell that is employed.Further, the vector must be able to accommodate the inserted nucleicacid sequence. Suitable host cells will include a wide variety ofeukaryotic or prokaryotic cells such as E. coli and the like.

The molecular weight of the antibodies of the invention will varydepending on several factors such as the intended use and whether theantibody includes a conjugated or recombinantly fused toxin,pharmaceutical, or detectable label or the like. Also the molecularweight will vary depending on nature and extent of post-translationalmodifications if any (such as glycosylation) to the antibody. Themodifications are a function of the host used for expression with E.coli producing non-glycosylated antibodies and mammalian cells producingglycosylated antibodies. In general, an antibody of the invention willhave a molecular weight of between approximately 20 to 150 kDa. Suchmolecular weights can be readily are determined by molecular sizingmethods such as SDS-PAGE gel electrophoresis followed by proteinstaining or Western blot analysis.

“Antibody of the invention” or other similar term refers to wholeimmunoglobulin as well as immunologically active fragments which bindnative TF. The immunoglobulins and immunologically active fragmentsthereof include an antibody-binding site (i.e., epitope capable of beingspecifically bound by an antibody recognizing native human TF capable ofspecifically binding native human TF). Exemplary antibody fragmentsinclude, for example, Fab, F(v), Fab′, F(ab′)₂ fragments, “halfmolecules” derived by reducing the disulfide bonds of immunoglobulins,single chain immunoglobulins, or other suitable antigen bindingfragments (see e.g., Bird et al., Science, pp. 242-424 (1988); Huston etal., PNAS, (USA), 85:5879 (1988); Webber et al., Mol. Immunol., 32:249(1995)). The antibody or immunologically active fragment thereof may beof animal (e.g., a rodent such as a mouse or a rat), or chimeric form(see Morrison et al., PNAS, 81:6851 (1984); Jones et al., Nature, pp.321, 522 (1986)). Single chain antibodies of the invention can bepreferred.

Similarly, a “nucleic acid of the invention” refers to a nucleotidesequence which can be expressed to provide an antibody of the inventionas such term is specified to mean immediately above.

As discussed above, antibodies of the invention can be administered to amammal, preferably a primate such as a human, to prevent or reducethrombotic occlusive disorders attributable to TF-mediated activation ofcoagulation, typically in a composition including one or morepharmaceutically acceptable non-toxic carriers such as sterile water orsaline, glycols such as polyethylene glycol, oils of vegetable origin,and the like. In particular, biocompatible, biodegradable lactidepolymer, lactide glycolide copolymer or polyoxyethylene,polyoxypropylene copolymers may be useful excipients to control therelease of the antibody-containing compositions described herein. Otherpotentially useful administration systems include ethylene vinyl acetatecopolymer particles, osmotic pumps, and implantable infusion systems andliposomes. Generally, an anti-coagulant composition of the inventionwill be in the form of a solution or suspension (or a lyophilized formthat can be reconstituted to a solution or suspension), and willpreferably include approximately 0.01% to 10% (w/w) of the antibody ofthe present invention, preferably approximately 0.01% to 5% (w/w) of theantibody. The antibody can be administered as a sole active ingredientin the composition, or as a cocktail including one or more otheranti-coagulant (e.g., heparin, hirudin or hirulog, coumadin, warfarin),anti-platelet (e.g., aspirin, Plavix, Ticlid, ReoPro, Integrilin orAggrestat), or thrombolytic agents (e.g., tissue plasminogen activator,strepokinase and urokinase). Additionally, antibodies of the inventioncan be administered prior to, or after administration of one or moresuitable anti-coagulant, anti-platelet or thrombolytic agents to boostor prolong desired anti-coagulation activity.

As also discussed above, antibodies of the invention can be employed toreduce potential blood coagulation arising from use of medicalimplementation, e.g. an indwelling device such as a catheter, stent,etc. In one preferred method, the implementation can be treated with anantibody of the invention (e.g., as a 1 mg/ml saline solution) prior tocontact with a body fluid. Alternatively, or in addition, an antibody ofthe invention can be combined with the body fluid in an amountsufficient to minimize blood clotting.

Therapeutic anti-coagulant compositions according to the invention aresuitable for use in parenteral or intravenous administration,particularly in the form of liquid solutions. Such compositions may beconveniently administered in unit dose and may be prepared in accordancewith methods known in the pharmaceutical art. See Remington'sPharmaceutical Sciences, (Mack Publishing Co., Easton Pa., (1980)). Bythe term “unit dose” is meant a therapeutic composition of the presentinvention employed in a physically discrete unit suitable as unitarydosages for a primate such as a human, each unit containing apre-determined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent orcarrier. The unit dose will depend on a variety of factors including thetype and severity of thrombosis to be treated, capacity of the subject'sblood coagulation system to utilize the antibody, and degree ofinhibition or neutralization of FX (or FIX) activation desired. Preciseamounts of the antibody to be administered typically will be guided byjudgment of the practitioner, however, the unit dose will generallydepend on the route of administration and be in the range of 10 ng/kgbody weight to 50 mg/kg body weight per day, more typically in the rangeof 100 ng/kg body weight to about 10 mg/kg body weight per day. Suitableregimens for initial administration in booster shots are also variablebut are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous or intermittent intravenousinfusions may be made sufficient to maintain concentrations of at leastfrom about 10 nanomolar to 10 micromolar of the antibody in the blood.

In some instances, it may be desirable to modify the antibody of thepresent invention to impart a desirable biological, chemical or physicalproperty thereto. More particularly, it may be useful to conjugate (i.e.covalently link) the antibody to a pharmaceutical agent, e.g. afibrinolytic drug such as t-PA, streptokinase, or urokinase to providefibrinolytic activity or to a targeting agent such as a fibrin-bindingdomain. Such linkage can be accomplished by several methods includinguse of a linking molecule such as a heterobifunctional proteincross-linking agent, e.g. SPDP, carbodimide, or the like, or byrecombinant methods.

In addition to pharmaceuticals such as a fibrinolytic agent, an antibodyof the invention can be conjugated to a toxin of e.g. plant or bacterialorigin such as diphtheria toxin (i.e., DT), shiga toxin, abrin, choleratoxin, ricin, saporin, pseudomonas exotoxin (PE), pokeweed antiviralprotein, or gelonin. Biologically active fragments of such toxins arewell known in the art and include, e.g., DT A chain and ricin A chain.The toxin can also be an agent active at cell surfaces such asphospholipases (e.g., phospholipase C). As another example, the toxincan be a chemotherapeutic drug such as, e.g., vendesine, vincristine,vinblastin, methotrexate, adriamycin, doxirubicin, bleomycin, orcisplatin, or, the toxin can be a radionuclide such as, e.g., iodine-131, yttrium-90, rhenium-1 88 or bismuth-212 (see generally, Moskaug etal., J. Biol. Chem., 264:15709 (1989); I. Pastan et al., Cell, 47:641(1986); Pastan et al., Recombinant Toxins as Novel Therapeutic Agents,Ann. Rev. Biochem., 61:331 (1992); Chimeric Toxins Olsnes and Phil,Pharmac. Ther., 25:355 (1982); published PCT Application No. WO94/29350; published PCT Application No. WO 94/04689; and U.S. Pat. No.5,620,939). Also, as discussed above, in addition to a toxin, anantibody of the invention can be conjugated to an effector molecule(e.g. IgG1 or IgG3) to provide complement-fixing ability andantibody-dependent cell-mediated cytoxicity upon administration to amammal.

Such an antibody-cytotoxin or effector molecule conjugate can beadministered in a therapeutically effective amount to a mammal,preferably a primate such as a human, where the mammal is known to haveor is suspected of having tumor cells, immune system cells, orendothelia capable of expressing TF. Exemplary of such tumor cells,immune system cells and endothelia include malignancies of the breastand lung, monocytes and vascular endothelia.

Antibodies of the invention also can be conjugated to a variety of otherpharmaceutical agents in addition to those described above such as,e.g., drugs, enzymes, hormones, chelating agents capable of binding aradionuclide, as well as other proteins and polypeptides useful fordiagnosis or treatment of disease. For diagnostic purposes, the antibodyof the present invention can be used either detectably labeled orunlabeled. For example, a wide variety of labels may be suitablyemployed to detectably-label the antibody, such as radionuclides,fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,ligands such as, e.g., haptens, and the like.

Diagnostic methods are also provided including in vivo diagnosticimaging [see, e.g., A. K. Abbas, Cellular and Molecular Immunology, pg.328 (W.B. Saunders Co. 1991)]. For most in vivo imaging applications, anantibody of the invention can be detectably-labeled with, e.g., ¹²⁵I,³²P, ⁹⁹Tc, or other detectable tag, and subsequently administered to amammal, particularly a human, for a pre-determined amount of timesufficient to allow the antibody to contact a desired target. Thesubject is then scanned by known procedures such as scintigraphic cameraanalysis to detect binding of the antibody. The analysis could aid inthe diagnosis and treatment of a number of thromboses such as thosespecifically disclosed herein. The method is particularly useful whenemployed in conjunction with cardiac surgery, particularly angioplasty,or other surgical procedure where undesired formation of a blood clotcan occur, to visualize the development or movement of a blood clot.

Antibodies of the invention also can be used to prepare substantiallypure (e.g., at least about 90% pure, preferably at least about 96 or 97%pure) native TF, particularly native human TF from a biological sample.For example, native TF can be obtained as previously described (seee.g., L. V. M. Rao et al., Thrombosis Res., 56:109 (1989)) and purifiedby admixing the solution with a solid support comprising the antibody toform a coupling reaction admixture. Exemplary solid supports include awall of a plate such as a microtiter plate, as well as supportsincluding or consisting of polystyrene, polyvinylchloride, across-linked dextran such as Sephadex™ (Pharmacia Fine Chemicals),agarose, polystyrene beads (Abbott Laboratories), polyvinyl chloride,polystyrene, polyacrylmide in cross-linked form, nitrocellulose or nylonand the like. The TF can then be isolated from the solid support insubstantially pure form in accordance with standard immunologicaltechniques. See generally Harlow and Lane supra and Ausubel et al.supra).

As also discussed above, antibodies of the invention can be employed todetect native human TF in a biological sample, particularly native TFassociated with a blood clot. Exemplary biological samples include bloodplasma, serum, saliva, urine, stool, vaginal secretions, bile, lymph,ocular humors, cerebrospinal fluid, cell culture media, and tissue,particularly vascular tissues such as cardiac tissue. Samples may besuitably obtained from a mammal suffering from or suspected of sufferingfrom a thrombosis, preferably restenosis, associated with, e.g., aninvasive medical procedure such as percutanous transluminal coronaryintervention, cardiopulmonary bypass surgery, endarterectomy, peripheralvascular bypass grafts, reconstructive or plastic surgery, jointreplacement; a heart ailment such as myocardial infarction,cardiomyopathy, valvular heart disease, stable angina, unstable angina,or artrial fibrillation associated with embolization; a coagulopathyincluding disseminated intravascular coagulation, deep vein thrombosis,deployment of an implementation such as a stent or catheter; shock(e.g., septic shock syndrome), vascular trauma, liver disease,hemorrhagic stroke, heat stroke, malignancies (e.g., pancreatic,ovarian, or small lung cell carcinoma), lupus, eclampsia, perivascularocclusive disease, and renal disease.

For such assays, an antibody of the invention can be detectably-labeledwith a suitable atom or molecule e.g., radioactive iodine, tritium,biotin, or reagent capable of generating a detectable product such as ananti-iodiotypic antibody attached to an enzyme such as P-galactosidaseor horseradish peroxidase, or a fluorescent tag (e.g., fluorescein orrhodamine) in accordance with known methods. After contacting thebiological sample with the detectably-labeled antibody, any unreactedantibody can be separated from the biological sample, the label (orproduct) is detected by conventional immunological methods includingantibody capture assay, antibody sandwich assay, RIA, ELISA,immunoprecipitation, immunoabsorption and the like (see Harlow and Lane,supra; Ausubel et al. supra). Any label (or product) in excess of thatdetected in a suitable control sample is indicative of the presence ofnative TF, more particularly a blood clot, in the biological sample. Forexample, antibodies of the invention can be detectably labeled todetect, and preferably quantitate, native TF in accordance with standardimmunological techniques such as antibody capture assay, ELISA, antibodysandwich assay, RIA, immunoprecipitation, immunoabsorption and the like.In some cases, particularly when a tissue is used, the immunologicaltechnique may include tissue fixation with a reagent known tosubstantially maintain protein conformation (e.g., dilute formaldehyde).See generally, Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, New York, (1989); Harlow and Lane in Antibodies: ALaboratory Manual, CSH Publications, NY (1988).

Antibodies of the invention also can be used for detecting and purifyingcells which express native TF, including fibroblasts, brain cells,immune cells, (e.g., monocytes), epithelia, as well as certain malignantcells. Preferred methods of detecting and purifying the cells includeconventional immunological methods (e.g., flow cytometry methods such asFACS, and immunopanning). Substantially pure populations of cellsexpressing native TF are useful in clinical and research settings, e.g.,to establish such cells as cultured cells for screening TF-bindingantibodies.

The invention also provides test and diagnostic kits for detection ofnative TF, particularly native human TF, in a test sample, especially abody fluid such as blood, plasma, etc., or tissue as discussed above. Apreferred kit includes a detectably labeled antibody of the invention.The diagnostic kit can be used in any acceptable immunological formatsuch as an ELISA format to detect the presence or quantity of native TFin the biological sample.

As discussed, the invention also features humanized antibodies thatspecifically bind to human tissue factor to form a binding complex. Thetissue factor may be naturally-occurring or recombinant (rHTF).Preferably, factor X or factor IX binding to the complex is inhibited.In a preferred invention embodiment, the humanized antibody has anaffinity constant (Kd) for the hTF of less than about 1 nM, preferablyless than about 0.5 nM, more preferably between from about 0.01 nM toabout 0.4 nM. See Example 11, below for more information aboutdetermining affinity constants for the humanized antibodies. By thephrase “specific binding” is meant that the humanized antibodies form adetectable binding complex with the TF and no other antigen asdetermined by standard immunological techniques such as RIA, Westernblot or ELISA.

Additional humanized antibodies of the invention are furthercharacterized by capacity to increase blood clotting time by at leastabout 5 seconds as determined by a standard prothrombin (PT) clottingassay. In preferred embodiments, the amount of humanized antibody willbe between from about 5 nM to about 75 nM, more preferably about 10 nMto about 50 nM, in the assay. See Example 11 below (describing how toperform the standard PT clotting assay with the humanized antibodies),for instance.

Additionally preferred humanized antibodies in accord with the inventionhave a binding specificity for tissue factor, preferably human TF, thatis about equal or greater than the antibody obtained from H36.D2.B7deposited under ATCC Accession No. HB-12255. Also preferred arehumanized antibodies which have a binding affinity for the TF aboutequal to or greater than the antibody obtained from H36.D2.B7 depositedunder ATCC Accession No. HB-12255. Methods for determining bindingspecificity and affinity are known in the field and include the specificassays described below.

Further humanized antibodies in accord with the invention include atleast one murine complimentarily determining region (CDR). As will beappreciated, immunoglobin light and heavy chain share certain structuralsimilarities eg., each includes a framework of four regions (FR1-4)whose sequences are relatively conserved. Each of FR1-4 (FR1, FR2, FR3,FR4) are covalently connected by three CDRs i.e., CDR1, CDR2, CDR3.There is general recognition that the four FRs largely adopt abeta-sheet configuration and the interconnected CDRs form loopsconnecting, and in some instances, forming part of the beta-sheetstructure. Most CDRs are held close to adjoining FRs, and with acorresponding CDR from the opposite light or heavy chain, help form theantigen binding site. A wide range of CDRs and FRs have been disclosed.See eg., Kabat et al. in Sequences of Proteins of Immunological InterestUS Dept. of Health and Human Services, US Government Printing Office(1987).

See also EP-A-0239400 and U.S. Pat. No. 5,985,279 (describing methods ofmaking altered antibodies in which CDRs are derived from differentspecies than the FR).

By the phrase “humanized” is meant an immunoglobin that includes a humanframework region and one or more CDRs from a non-human source, usuallyrodent such as a rat or mouse immunoglobin. The non-human immunoglobinproviding the CDRs is called a “donor” and the human immunoglobin calledthe “acceptor”. Constant regions need not be present, as in, forexample, certain TF binding fragments of such immunoglobins. Preferredconstant regions, if present, are substantially identical to humanimmunoglobin constant regions i.e., at least about 90% identical withregard to the amino acid sequence, preferably at least about 95%identical or greater. Accordingly, nearly all parts of the humanizedimmunoglobin, with the possible exception of the CDRs are substantiallyidentical to corresponding parts of naturally-occurring humanimmunoglobin sequences.

By the phrase “humanized antibody” is meant an antibody that includes ahumanized light chain and a humanized heavy chain immunoglobin. Methodsfor making and using such antibodies have already been discussed above.See S. L. Morrison, supra; Oi et al., supra; Teng et al., supra; Kozboret al., supra; Olsson et al.,supra; and other references citedpreviously.

For example, an illustrative humanized antibody includes: 1) light andheavy chain frameworks (FRs) that are each at least about 90% identicalin amino acid sequence, preferably at least 95% identical tocorresponding human FRs, 2) at least one CDR from a mouse, preferablyall the CDRs from the mouse, 3) and an immunoglobin constant region thatis at least about 90% identical, preferably at least 95% identical to acorresponding human immunoglobin constant region. It will be appreciatedthat the donor antibody has been “humanized” by the process of“humanization” because the resultant humanized antibody is expected tobind to the same antigen as the donor antibody that provides the CDRs.

It will be further appreciated that the humanized antibodies providedherein may have one or more additional conservative amino acidsubstitutions which can be contiguous or non-contiguous as needed. Forexample, such substitutions will typically have substantially little orno effect on antigen binding or other immunoglobin functions. By thephrase “conservative substitution” including plural forms is meantcombinations of: gly

ala; val

ile

leu; asp

glu; asn

gln; ser

thr, lys

arg; and phe

tyr.

Additional humanized antibodies feature a variable region that is atleast 70% identical in amino acid sequence (eg., about 73% to 75%identical), to the corresponding variable region of one or more nativehuman immunoglobin sequences. Further humanized antibodies in accordwith the invention have at least 90% identity over the entire antibodyto one or more human antibodies.

More specific humanized antibodies of the invention are those in each offrameworks (FRs) 1, 2, 3 and 4 has at least about 90% amino acidsequence identity, preferably at least about 95% or greater identity tothe light chain FR sequences shown in FIG. 12A (SEQ ID NO. 72-82,respectively, in order of appearance). Preferably, the sequence is asshown as “LC-09” in FIG. 12A (SEQ ID NO: 79). Further preferred arethose humanized antibodies that include a light chain constant regionhaving at least about 90% amino acid sequence identity, and preferably,at least about 95% sequence identity or greater to the sequence shown inFIG. 14A or 15A (SEQ ID NOS: 97 and 99, respectively).

Further specific humanized antibodies are those in which each offrameworks (FRs) 1, 2, 3 and 4 has at least about 90% amino acidsequence identity, preferably about 95% identity or greater to the heavychain sequences shown in FIG. 13A (SEQ ID NOS: 83-96, respectively, inorder of appearance). Preferably, the sequence is as shown as “HC-08”(SEQ ID NO: 91) in FIG. 13A. Additional humanized antibodies have aheavy chain constant region with at least about 90% amino acid sequenceidentity, and preferably, at least about 95% identity or greater, tosequence shown in FIG. 14B or 15B (SEQ ID NOS: 98 and 100,respectively).

In certain embodiments, the humanized antibody will have an IgG1 (hOAT)or IgG4 (hFAT) isotype. See Example 9.

Also provided by the present invention are functional fragments of thehumanized antibodies disclosed herein. Preferred fragments specificallybind TF with an affinity constant (Kd) of less than about 1 nM,preferably less than about 0.5 nM, more preferably between from about0.01 nM to about 0.4 nM. Specifically preferred are antigen binding Fab,Fab′, and F(ab)₂ fragments.

As discussed, the invention features humanized antibodies that includeat least one murine complementarity determining region (CDR), eg., CDR1,CDR2, CDR3. In a preferred embodiment, the antibodies bind specificallyto human tissue factor (TF) to form a complex. Typically, the factor Xor factor IX binding to TF or TF:VIIa and activation by TF:FVIIa theretois inhibited. As mentioned above, preferred CDRs (light and heavy chain)are from a rodent source, typically the mouse.

In one embodiment of the humanized antibodies of the invention, theantibodies further include at least one human framework (FR) region.Preferably, all the FR regions (light and heavy chain) are human.

In a more particular embodiment, the first CDR (CDR1) of the heavy chainhypervariable region is at least 90% identical to the CDR1 amino acidsequences shown in FIG. 13B (both SEQ ID NO: 8), preferably at leastabout 95% identical or greater to that sequence. Typically, the secondCDR (CDR2) of the heavy chain hypervariable region is at least 90%identical to the CDR2 amino acid sequence shown in FIG. 13C (SEQ ID NOS:9 and 101), preferably at least about 95% identical or greater.Preferably also, the third CDR (CDR3) of the heavy chain hypervariableregion is at least 90% identical to the CDR3 sequence shown in FIG. 13D(both SEQ ID NO: 10), more preferably about 95% identical or greater tothat sequence.

Identity between two nucleic acid sequences can be determined byinspection and/or use of conventional computer software such as BLASTand FASTA.

In another invention embodiment, the first CDR (CDR1) of the light chainhypervariable region is at least 90% identical to the CDR1 amino acidsequence shown in FIG. 12B (fragment of SEQ ID NO: 2), preferably atleast about 95% identical or greater. Typically, the second CDR (CDR2)of the light chain hypervariable region is at least 90% identical to theCDR2 amino acid sequence shown in FIG. 12C (SEQ ID NO: 6), preferablyabout 95% identical or greater. Preferably, the third CDR (CDR3) of thelight chain hypervariable region is at least 90% identical to the CDR3amino acid sequence shown in FIG. 12D (SEQ ID NO: 7), more preferablyabout 95% identical or greater to that sequence.

Additional humanized antibodies of the invention include a firstframework (FR1) of the heavy chain hypervariable region which FR1 is atleast 90% identical to the FRi amino acid sequence shown in FIG. 13A(fragment of SEQ ID NO: 91) as “FR1 HC-08”, preferably about 95%identical or greater to that sequence. In one embodiment, the FR1comprises at least one of the following amino acid changes: E1 to Q; Q5to V; P9 to G; L11 to V; V12 to K; Q19 to R; and T24 to A. Preferably,the FR1 includes two, three, four, five, or six of those changes withall of those amino acid changes being preferred for many applications.

Further humanized antibodies of the invention include a second framework(FR2) of the heavy chain hypervariable region which FR2 is at least 90%identical to the FR2 sequence shown in FIG. 13A (fragment of SEQ ID NO:91) as “FR2 HC-08”, preferably about 95% identical or greater to thatsequence. In one embodiment, the FR2 at least one of the following aminoacid changes: 41H to P; and 44S to G. A preferred FR2 includes both ofthose amino acid changes.

The invention also features humanized antibodies in which a thirdframework (FR3) of the heavy chain hypervariable region is at least 90%identical to the FR3 sequence shown in FIG. 13A (fragment of SEQ ID NO:91) as “FR3 HC-08”, preferably about 95% identical or greater to thatsequence. In one embodiment, the FR3 includes at least one of thefollowing amino acid changes: 76S to T; 77T to S; 80F to Y; 82H to E;84N to S; 87T to R; 89D to E; and 91S to T. A preferred FR3 includestwo, three, four, five or six of those amino acid changes with all sevenof those amino acid changes being generally preferred.

Also featured are humanized antibodies in which the fourth framework(FR4) of the heavy chain hypervariable region is at least 90% identicalto the FR4 amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO:91) as “FR4 HC-08”, preferably at least about 95% identical or greaterto that sequence. Preferably, the FR4 includes the following amino acidchange: 113L to V.

Additional humanized antibodies in accord with the invention feature afirst framework (FR1) of the light chain hypervariable region which isat least about 90% identical to the FR1 amino acid sequence shown inFIG. 12A (fragment of SEQ ID NO: 79) as “FR1 LC-09”, preferably at leastabout 95% identical or greater to that sequence. In one embodiment, theFR1 comprises at least one of the following amino acid changes: 11Q toL; 15L to V; 17E to D; and 18 to R. A preferred FR1 includes two orthree of such amino acid changes with all four amino acid changes beinggenerally preferred.

The present invention also features humanized antibodies in which asecond framework (FR2) of the light chain hypervariable region is atleast about 90% identical to the FR2 amino acid sequence shown in FIG.12A (fragment SEQ ID NO: 79) as “FR2 LC-09”, preferably at least about95% identical or greater to that sequence. A preferred FR2 has thefollowing amino acid change: 37Q to L.

Also encompassed by the invention are humanized antibodies in which athird framework (FR3) of the light chain hypervariable region is atleast about 90% identical to the FR3 amino acid sequence shown in FIG.12A (fragment of SEQ ID NO: 79) as “FR3 LC-09 ”, preferably at leastabout 95% identical or greater to that sequence. In one embodiment, theFR3 has at least one of the following amino acid changes: 70K to D, 74Kto T, 80A to P, 84A to V, and 85N to T. Preferably, the FR3 has two,three, or four of such amino acid changes with all five of the changesbeing generally preferred.

Additional humanized antibodies of the invention include a fourthframework (FR4) of the light chain hypervariable region which FR4 is atleast about 90% identical to the sequence shown in FIG. 12A (fragment ofSEQ ID NO: 79) as “FR4 LC-09”, preferably at least about 95% identicalor greater to that sequence. In one embodiment, the FR4 includes atleast one and preferably all of the following amino acid changes: 100Ato Q; and 106L to I.

The invention also features a human TF binding fragment of the foregoinghumanized antibodies. Examples of such fragments include Fab, Fab′, andF(ab)₂.

In a particular embodiment, the invention features a humanized antibodythat includes at least one rodent complementarity determining region(CDR), usually mouse. Preferably, that antibody binds specifically tohuman tissue factor (TF) to form a complex in which factor X or factorIX binding to TF or TF/VIIa and activation by TF/VIIa thereto isinhibited. Also preferably, the humanized antibody includes, on theheavy chain, at least one of and more preferably all of the followingcomponents:

a) a first CDR (CDR1) which is at least 95% identical to CDRl amino acidsequences shown in FIG. 13B (SEQ ID NO: 8),

b) a second CDR (CDR2) which is at least 95% identical to the CDR2 aminoacid sequence shown in FIG. 13C (SEQ ID NO: 9 or 101),

c) a third CDR (CDR3) which is at least 95% identical to the CDR3 aminoacid sequence shown in FIG. 13D (SEQ ID NO: 10),

d) a first framework (FR 1) which is at least 95% identical to the FR1amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO: 91) as“FR1 HC-08”,

e) a second framework (FR2) which is at least 95% identical to the FR2amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO: 91) as“FR2 HC-08”,

f) a third framework (FR3) which is at least 95% identical to the FR3amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO: 91) as“FR3 HC-08”, and

g) a fourth framework (FR4) which is at least 95% identical to the FR4amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO: 91) as“FR4 HC-08”.

In a particular embodiment, the humanized antibody also includes, on thelight chain, at least one of and preferably all of the followingcomponents:

h) a first CDR (CDR1) which is at least 95% identical to CDR1 amino acidsequence shown in FIG. 12B (fragment of SEQ ID NO: 2),

i) a second CDR (CDR2) which is at least 95% identical to the CDR2 aminoacid sequence shown in FIG. 12C (SEQ ID NO: 6),

j) a third CDR (CDR3) which is at least 95% identical to the CDR3 aminoacid sequence shown in FIG. 12C (SEQ ID NO: 6),

k) a first framework (FR 1) which is at least 95% identical to the FR1amino acid sequence shown in FIG. 12A (fragment of SEQ ID NO: 79) as“FR1 LC-09”,

l) a second framework (FR2) which is at least 95% identical to the FR2amino acid sequence shown in FIG. 12A (fragment of SEQ ID NO: 79) as“FR2 LC-09”,

m) a third framework (FR3) which is at least 95% identical to the FR3amino acid sequence shown in FIG. 12A (fragment of SEQ ID NO: 79) as“FR3 LC-09”, and

n) a fourth framework (FR4) which is at least 95% identical to the FR4amino acid sequence shown in FIG. 12A (fragment of SEQ ID NO: 79) as“FR4 LC-09”. Preferably, the humanized antibody further includes thelight chain constant sequence of FIG. 14A (SEQ ID NO: 97) or FIG. 15A(SEQ ID NO: 99). Also preferably, the antibody includes the heavy chainconstant region of FIG. 14B (SEQ ID NO: 98) or FIG. 15B (SEQ ID NO:100).

The invention also features a humanized antibody that includes, on theheavy chain, at least one of and preferably all of the followingcomponents:

a) a first CDR (CDR1) identical to the CDR1 amino acid sequence shown inFIG. 13B (SEQ ID NO: 8),

b) a second CDR (CDR2) identical to the CDR2 amino acid sequence shownin FIG. 13C (SEQ ID NOS: 9 or 101),

c) a third CDR (CDR3) identical to the CDR3 amino acid sequence shown inFIG. 13D (SEQ ID NO: 10),

d) a first framework (FR1) identical to the FR1 amino acid sequenceshown in FIG. 13A (fragment of SEQ ID NO: 91) as “FR1 HC-08”,

e) a second framework (FR2) identical to the FR2 amino acid sequenceshown in FIG. 13A (fragment of SEQ ID NO: 91) as “FR2 HC-08”,

f) a third framework (FR3) identical to the FR3 amino acid sequenceshown in FIG. 13A (fragment of SEQ ID NO: 91) as “FR3 HC-08”; and

g) a fourth framework (FR4) identical to the FR4 amino acid sequenceshown in FIG. 13A (fragment of SEQ ID NO: 91) as “FR4 HC-08”.

In one embodiment, the humanized antibody further includes, on the lightchain, at least one of and preferably all of the following components:

h) a first CDR (CDR1) identical to CDR1 amino acid sequence shown inFIG. 12B (fragment SEQ ID NO: 2),

i) a second CDR (CDR2) identical to the CDR2 amino acid sequence shownin FIG. 12C (SEQ ID NO: 6),

j) a third CDR (CDR3) identical to the CDR3 amino acid sequence shown inFIG. 12D (SEQ ID NO: 7),

k) a first framework (FRi) identical to the FR1 amino acid sequenceshown in FIG. 12A (fragment of SEQ ID NO: 79) as “FRi LC-09”,

l) a second framework (FR2) identical to the FR2 amino acid sequenceshown in FIG. 12A (fragment of SEQ ID NO: 79) as “FR2 LC-09”,

m) a third framework (FR3) identical to the FR3 amino acid sequenceshown in FIG. 12A (fragment of SEQ ID NO: 79) as “FR3 LC-09”, and

n) a fourth framework (FR4) identical to the FR4 amino acid sequenceshown in FIG. 12A (fragment of SEQ ID NO: 79) as “FR4 LC-09”.Preferably, the humanized antibody further includes the light chainconstant sequence of FIG. 14A (SEQ ID NO: 97) or FIG. 15A (SEQ ID NO:99). Also preferably, the antibody includes the heavy chain constantregion of FIG. 14B (SEQ ID NO: 98) or FIG. 15B (SEQ ID NO: 100).

The humanized antibodies of the present invention may exist in a varietyof suitable forms in addition to whole antibodies; including, forexample, Fv, Fab, and F(ab′)₂ as well as bifunctional hybrid antibodies(e.g., Lanzavecchia et al., Eur. J Immunol. 17, 105 (1987)) and insingle chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which areincorporated herein by reference). (See, Hood et al., Immunology,Benjamin, N.Y., 2.sup.nd ed. (1984), Harlow and Lane, Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapillerand Hood, Nature, 323, 15-16 (1986), which are incorporated herein byreference.).

By the phrase “chimeric antibody” or related phrase including pluralforms is meant antibodies whose light and heavy chain genes have beenconstructed, typically by genetic engineering, from immunoglobulin genesegments belonging to different species. For example, the variable (V)segments of the genes from a mouse monoclonal antibody may be joined tohuman constant (C) segments, such as γ₁ γ₃. A typical therapeuticchimeric antibody is thus a hybrid protein consisting of the V orantigen-binding domain from a mouse antibody and the C or effectordomain from a human antibody, although other mammalian species may beused. A specifically preferred chimeric antibody is the cH36 mouse-humanchimera disclosed herein.

The humanized antibodies of the present invention can be polyclonal ormonoclonal, as needed, and may have an IgG1 or IgG4 isotype.

The humanized antibodies disclosed herein can be produced by one or acombination of strategies including those already referenced above. Seeeg., S. L. Morrison, supra; Oi et al., supra; Teng et al., supra; Kozboret al., supra; Olsson et al.,supra; and other references citedpreviously.

In one approach, four general steps were employed to humanize theantibodies. First, the amino acid sequences of the mouse antibody lightand heavy chains were obtained from the cH36 mouse-human chimericantibody. Second, the cH36 antibody was humanized by determining whichhuman antibody framework region gave the “best fit” i.e., most closelyresembled the corresponding mouse framework amino acid sequence. Third,relevant light and heavy chain FR sequences were humanized, and fourth,transfection and expression of isolated nucleic acid(s) that encode thehumanized light or heavy chain (or humanized light and heavy chain e.g.,see the mega vectors described below).

In some instances, a limited number of framework amino acids of ahumanized immunoglobin were chosen to be the same as the amino acids atthose positions in the donor rather than in the acceptor. One advantageof this technique is to enhance affinity of the antibody that includesthe humanized immunoglobin chain. See also U.S. Pat. Nos. 5,985,279;5,693,762; and EP-A0239400 (disclosing general methods for makinghumanized antibodies).

More particularly, the “best fit” approach was applied to humanizing thechimeric anti-tissue factor antibody cH36 is specifically preferred. Inthis approach, the murine light and heavy chain variable sequences shownin FIGS. 1A and 1B (SEQ ID NOS: 2 and 4) were used to search (“compare”)all available protein databases for those human antibody variable domainsequences that are most homologous to the murine variable domain. Seee.g., Kabat et al., supra. A number of readily available computerprograms can be used to perform this step such as BLAST, FASTA andrelated programs. Frameworks 1, 2, 3, and 4 of the light and heavy chainwere of special interest since these sites are almost universallyunderstood to hold the CDRs in proper orientation for antigen binding.Output stemming from the search was typically a list of sequences mosthomologous to the query mouse sequences, the percent homology to eachsequence, and an alignment of each human sequence to the correspondingmurine sequence. The analysis was generally performed on the light andheavy chains independently.

According to the “best fit” approach, the number of mismatched aminoacids was minimized between the query mouse framework sequence and thecorresponding human framework sequence in the database. In most cases,suitable human framework regions were selected based on the followingidentity criteria. On the light chain, the amino acid sequence of themurine FR1 was at least about 80% identical to the corresponding humanFR1; the murine FR2 was at least about 90% identical to thecorresponding human FR2, the murine FR3 was at least about 90% identicalto the human FR3; and the murine FR4 was at least about 75% identical tothe corresponding human FR4. And on the heavy chain, the amino acidsequence of the murine FR1 was chosen to be at least about 80% identicalto the corresponding human FR1; the murine FR2 was at least about 85%identical to the human FR2; the murine FR3 was chosen to be at leastabout 70% identical to the corresponding human FR3; and the murine FR4was at least about 90% identical to the corresponding human FR4.Typically, conservative amino acid substitutions were favored whenevaluating similar candidate human framework sequences. It was foundthat when such factors were considered the resulting human frameworksserved as a good reference point for humanization of the chimeric cH36antibody.

Also preferably, according to the “best fit” approach all of the humanframeworks on the light and heavy chain were derived from the same humanantibody clone where possible.

Once a decision on a desired human framework was made, recombinantpolymerase chain reaction (PCR) techniques were used to make desiredamino acid substitutions in both the light and heavy chains. Typically,oligonucleotides were made and used to mutagenize mouse variable domainframeworks to contain desired residues. Oligonucleotides having avariety of lengths were employed. See WO 92/07075 for general disclosurerelating to recombinant PCR and related methods.

In general, regular PCR was used for cloning, to introduce cloning ordiagnostic endonuclease sites, and to change amino acid residues locatedat the ends of the variable regions. PCR-based mutagenesis was used tochange multiple amino acid residues at a time, especially when theseresidues were in the middle of the variable regions. Site directedmutagenesis was used to introduce one or two amino acid substitutions ata time. After each step, the partially humanized clones were sequencedand some of these variable regions were later cloned into expressionvectors. More specific methods for performing these manipulations aredescribed in the Examples section.

After performing the foregoing “best fit” approach to humanizing thechimeric cH36 antibody, mutagenized nucleic acids encoding frameworkand/or CDR were linked to an appropriate DNA encoding a light or heavychain constant region. Such constructs were then cloned into anexpression vector, and transfected into host cells, preferably mammaliancells. These steps were achieved by using recombinant and cell culturetechniques known in the field. Accordingly, a humanized antibody of theinvention can be prepared by the following general method:

(a) preparing a first expression vector including a replicon appropriatefor the expression host and a suitable promoter operably linked to a DNAsequence which encodes at least a variable domain of an Ig heavy orlight chain, the variable domain comprising humanized framework regions1-4 made according to the “best fit” approach and murine CDRs 1-3 fromthe cH36 antibody,

(b) preparing a second replicable expression vector including a suitablepromoter operably linked to a DNA sequence which encodes at least thevariable domain of a complementary Ig light or heavy chain respectively,that variable domain comprising complementary humanized frameworkregions 1-4 made according to the foregoing “best fit” approach andmurine CDRs 1-3 from the cH36 antibody;

(c) transfecting a cell line with the first or both prepared vectors;and

(d) culturing said transfected cell line to produce said alteredantibody.

Preferably the DNA sequence in steps (a) and (b) encode suitableconstant domains from the human antibody chain. Suitable isotypesinclude IgG1 and IgG4, for example.

Alternatively, a suitable humanized antibody of the invention can beprepared by making a single replicable “mega” vector that includes anappropriate promoter operably linked to a DNA sequence which encodes atleast a variable domain of an Ig heavy or light chain, the variabledomain comprising humanized framework regions 1-4 made according to the“best fit” approach and murine CDRs 1-3 from the cH36 antibody.Preferably, the mega vector will further include a suitable promoteroperably linked to a DNA sequence which encodes at least the variabledomain of a complementary Ig light or heavy chain respectively, thatvariable domain comprising complementary humanized framework regions 1-4made according to the foregoing “best fit” approach and murine CDRs 1-3from the cH36 antibody. Use of the mega vector will often be appropriatein invention embodiments in which humanized antibody expression from asingle vector is needed.

Other methods are well-suited for making the humanized antibodies andfragments of this invention. In one embodiment, the method includes atleast one and preferably all of the following steps:

a) comparing the amino acid sequence of a light chain framework from arodent antibody against a collection of corresponding human antibodyframework sequences, preferably a mouse antibody,

b) selecting a human framework sequence from the collection having thegreatest amino acid sequence identity (i.e., at least about 70% sequenceidentity) to the corresponding rodent light chain framework,

c) mutagenizing a DNA segment encoding the rodent light chain frameworkto encode a humanized light chain framework having an amino acidsequence that is substantially identical (i.e., at least about 95%identical) to the human framework sequence selected in step b),

d) repeating steps a) thru c) for each individual framework of therodent light chain to produce a plurality of DNA sequences in which eachsequence encodes a humanized light chain framework in which each of thecorresponding human framework sequences selected in step b) ispreferably from the same or different human antibody,

e) assembling into a first vector encoding at least the light chainvariable region of the rodent antibody, the DNA sequences encoding thehumanized framework sequences produced in step d); and

f) introducing the assembled vector into a suitable host underconditions sufficient to produce the humanized antibody. Preferred lightchain framework sequences for use with the method include those specificmouse and humanized light chain frameworks disclosed herein.

In one embodiment, the foregoing method for making the humanizedantibody further includes at least one and preferably all of thefollowing steps:

g) comparing the amino acid sequence of a heavy chain framework from therodent antibody against a collection of corresponding human antibodyframework sequences,

h) selecting a human framework sequence from the collection having thegreatest amino acid sequence identity (i.e., at least about 70% sequenceidentity) to the corresponding rodent heavy chain framework,

i) mutagenizing a DNA segment encoding the rodent heavy chain frameworkto encode a humanized heavy chain framework having an amino acidsequence that is substantially identical (i.e., at least about 95%identical) to the human framework sequence selected in step h); and

j) repeating steps g) thru i) for each individual framework of therodent heavy chain to produce a plurality of DNA sequences in which eachsequence encodes a humanized heavy chain framework. Preferably, each ofthe corresponding human framework sequences selected in step h) are fromthe same or different human antibody. Preferred heavy chain frameworksequences for use with the method include those specific mouse andhumanized heavy chain frameworks disclosed herein.

More particular methods for making the humanized antibody includeassembling into a second vector encoding at least the heavy chainvariable region of the rodent antibody, the DNA sequences encoding thehumanized framework sequences produced in step j); and introducing theassembled first and second vectors into the host under conditionssufficient to produce the humanized antibody.

As discussed, it will often be preferable to express the humanizedantibodies of this invention from a single vector which can sometimes bea “mega” vector. In one embodiment, the method includes assembling intothe first vector encoding at least the heavy chain variable region andthe light chain variable region of the rodent antibody, the DNAsequences encoding the humanized framework sequences produced in stepj); and introducing the further assembled first vector into the hostunder conditions sufficient to produce the humanized antibody.

By the words “assembling” or “assembled” is meant use of standardrecombinant techniques to introduce subject DNA sequences encoding thehumanized frameworks into the vectors. Such assembly can be performed byone or combination of approaches including, but not limited to,introducing iterative changes to a single framework sequence, cuttingand pasting fragments together (via use of restriction endonucleases andligase), or by synthetic DNA synthesis techniques. See generally Harlowand Lane supra and Ausubel et al. supra.

The foregoing methods for making humanized antibodies can be practicedwith nearly any acceptable mutagenesis technique. In particular, one orboth of steps c) and i), above, can employ site directed mutagenesis orstandard PCR methods to replace desired rodent amino acids in theframework with appropriate human amino acids. Typically, the sequence ofthe modified (humanized) framework corresponds to the selected humanframework sequence from the database.

The humanized antibody can be prepared using any suitable recombinantexpression system such as those disclosed in S. L. Morrison, supra; Oiet al., supra; Teng et al., supra; Kozbor et al., supra; Olsson etal.,supra; and other references cited previously.

For example, suitable nucleic acids of the invention encode at least oneof the heavy or light chain of the humanized antibodies or fragmentsthereof disclosed herein. Typically, the nucleic acid is a recombinantDNA vector that includes the isolated nucleic acid. The DNA vector willtypically further include an expression control polynucleotide sequenceoperably linked to the humanized immunoglobulin coding sequences,including naturally-associated or heterologous promoter regions.Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells, but control sequences for prokaryotic hosts may also beused. Once the vector has been incorporated into the appropriate host,the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the light chains, heavy chains, light/heavy chaindimers or intact antibodies, binding fragments or other immunoglobulinforms may follow.

The nucleic acid sequences of the present invention capable ofultimately expressing the desired humanized antibodies can be formedfrom a variety of different polynucleotides (genomic or cDNA, RNA,synthetic oligonucleotides, etc.) and components (e.g., V, J, D, and Cregions), as well as by a variety of different techniques. Joiningappropriate genomic and synthetic sequences is presently the most commonmethod of production, but cDNA sequences may also be utilized. See eg.,S. L. Morrison, supra; Oi et al., supra; Teng et al., supra; Kozbor etal., supra; Olsson et al.,supra; European Patent Publication No. 0239400and Riechmann, L. et al., Nature, 332, 323-327 (1988); and referencescited therein.

In one embodiment, suitable DNA expression vectors include one or moreselection markers, e.g., tetracycline, ampicillin, or neomycin, topermit detection of those cells transformed with the desired DNAsequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporatedherein by reference). E. coli is one prokaryotic host usefulparticularly for cloning the polynucleotides of the present invention.Other microbial hosts suitable for use include but are not limited tobacilli, such as Bacillus subtilus, and other enterobacteriacea, such asSalmonella, Serratia, various Pseudomonas species and other microbessuch as actinomycetes (e.g., Streptomyces species), yeast (e.g.,Saccharomyces species) or fungi (e.g., Aspergillus species). In theseprokaryotic hosts, one can also make expression vectors, which willtypically contain expression control sequences compatible with the hostcell (e.g., promoters and an origin of replication). In addition, anynumber of a variety of well-known promoters will be present, such as thelactose promoter system, a tryptophan (trp) promoter system, abeta-lactamase promoter system, or a promoter system from phage lambda.The promoters will typically control expression, optionally with anoperator sequence, and have ribosome binding site sequences and thelike, for initiating and completing transcription and translation. Othermicrobes, such as yeast, may also be used for expression. Saccharomycesis a preferred host, with suitable vectors having expression controlsequences, such as promoters, including 3-phosphoglycerate kinase orother glycolytic enzymes, and an origin of replication, terminationsequences and the like as desired. Plants (e.g., Arabidopsis, Nicotinia,etc.) and plant cell culture may also be used to express and produce theantibodies of the present invention In addition to forgoingmicroorganism-based systems, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(see, Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y.(1987), which is incorporated herein by reference).

In many instances, eukaryotic cells will be generally preferred,typically CHO cell lines, various COS cell lines, NSO cells, BK cells,HeLa cells, preferably myeloma cell lines, etc., or transformed B-cellsof hybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, andenhancer (Queen et al., Immunol. Rev. 89, 46-68 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus,cytomegalovirus and the like.

Preferred DNA vectors for practicing the invention include the followingoperatively linked sequences: an antibiotic resistance marker e.g.,ampicillin resistance, F1 origin, and heavy chain (HC) or light chain(LC) variable region. That variable region can be inserted into anappropriate HC expression that includes operatively linked in sequence:the HC variable region, human IgG1 or IgG4 constant region, first poly Asite, SV40 promoter, antibiotic resistance marker such as neomycinresistance, second poly A site, cytomegelovirus (CMV) promoter/enhancer,and suitable leader sequence.

Additionally preferred DNA vectors include the LC variable regionoperatively linked to a rodent kappa intron (e.g., mouse) which intronis operatively linked to a suitable human kappa constant region; andantibiotic resistance marker such a neomycin resistance.

As discussed, it will often be highly useful to express humanizedantibodies of the present invention from a single nucleic acid. Apreferred DNA vector is sometime referred to herein as a “mega” vectorand includes operatively linked in sequence the following components:SV40 promoter, antibiotic resistance marker such as neomycin, first polyA site, first CMV promoter/enhancer, LC variable region, rodent kappaintron (e.g., mouse), human kappa exon, second poly A site, second CMVpromoter/enhancer, HC variable sequence, and human IgG1 or IgG4 heavychain constant region. A specific example of such a mega vector is thehumanized anti-TF IgG1 antibody expression vector described below inExample 10. See also FIG. 11.

The following three nucleic acid vectors pSUN36 (humanized anti-TFantibody Ig G1-HC expression vector), pSUN37 (humanized anti-TF antibodyIg G4-HC expression vector), and pSUN38 (humanized anti-TF antibody LCexpression vector) have been deposited pursuant to the Budapest Treatywith the American Type Culture Collection (ATCC) at 10801 UniversityBoulevard, Manassas Va. 20110-2209. The vectors were assigned thefollowing Accession Numbers: PTA-3727 (pSUN36); PTA-3728 (pSUN37); andPTA-3729 (pSUN38).

A variety of suitable host cells can be used to produce the humanizedantibodies or fragments disclosed herein. In one embodiment the methodincludes providing a host cell transfected with either 1) a firstexpression vector encoding the light chain of the humanized antibody orfragment thereof and a second expression vector encoding the heavy chainof the humanized antibody or fragment thereof, or 2) a single expressionvector encoding both the light chain and the heavy chain of thehumanized antibody or fragment thereof, maintaining the host cell undergrowth conditions in which each chain is expressed; and isolating thehumanized antibody or fragment thereof.

For example, the cell line that is transfected to produce the humanizedantibody can be Chinese Hamster Ovary (CHO) cell line, BK cell line orNSO cell line. Further acceptable cell lines include recognizedimmortalized mammalian cell lines, preferably of lymphoid origin, suchas a myeloma, hybridoma, trioma or quadroma cell lines. The cell linemay also comprise a normal lymphoid cell, such as a B-cell, which hasbeen immortalized by transformation with a virus, such as theEpstein-Barr virus. Methods for using CHO cells for expression of avariety of proteins have been reported. See e.g., Urlaub et al., Proc.Natl. Acad. Sci. U.S.A., 77 4216-4220 (1980)) and WO 87/04462. NSOcells, as described below in the Examples section, are also preferred.

Although the cell line used to produce the humanized antibody ispreferably a mammalian cell line, any other suitable cell, such as abacterial cells, plant cells, insect cells or yeast cells, mayalternatively be used. In particular, it is envisaged that E.coli-derived bacterial strains could be used.

Once expressed from an appropriate cell source, the whole antibodies,their dimers, individual light and heavy chains, or other immunoglobulinforms of the present invention such as functional humanized antibodyfragments can be recovered and purified according to standardprocedures. Such procedures include, but are not limited to, ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like (See, generally, Scopes, R., ProteinPurification, Springer-Verlag, N.Y. (1982)). Substantially purehumanized antibodies of the invention and fragments thereof feature atleast about 90 to 95% homogeneity with about 98 to 99% or morehomogeneity being generally preferred for most pharmaceutical uses. Oncepurified, partially or to homogeneity as desired, a humanized antibodymay then be used therapeutically or in developing and performing assayprocedures, immunofluorescent stainings, and the like (See, generally,Immunological Methods, Vols. I and II, Lefkovits and Pemis, eds.,Academic Press, New York, N.Y. (1979 and 1981)).

A preferred method of purifying the present humanized antibodiesinvolves conventional affinity and ion exchange chromatography,preferably using recombinant Protein A Sepharose (to which human Ig G Fchas recognized high affinity). Antibody containing fractions arecollected and subjected to further ion exchange chromatography,preferably using Q Sepharose. Antibody containing protein peaks arepooled and dialyzed against an appropriate solution or buffer, forinstance, PBS.

Humanized antibodies and fragments thereof according to the inventioncan be tested for function by one or a combination of standard methods.Preferred tests assay for inhibition of TF function. A preferred methodis what is sometimes referred to herein as a “standard prothrombin time”assay or related phrase. The standard prothrombin time (PT) assaytypically involves at least one and preferably all of the followingsteps:

-   -   a) combining TF and factor VIIa to form a binding complex,    -   b) contacting the binding complex with factor X (or factor IX)        under conditions conducive to forming factor Xa (or factor IXa),    -   c) contacting the factor Xa with prothrombin to produce        thrombin, preferably in the presence of factor Va and lipids.

A preferred source of the TF for conducting the standard PT assay iscommercially available as Innovin. A preferred source for the bloodfactors is a human plasma preparation called Ci-Trol CoagulationControl.

The humanized antibodies and fragments thereof provided herein can bereadily tested in the assay. An aliquot of the purified antibody orfragment, preferably about 200 nM to about 2000 nM, is added to themethod, preferably before step a) although addition at other points inthe assay may be preferred for some applications. Typically, thehumanized antibody or fragment is added to the Ci-Trol CoagulationControl followed by addition of the TF.

Highly preferred humanized antibodies and fragments thereof includingwhole IgG, Fab, Fab′, F(ab)₂, and single chain antibodies (comprisingthe antigen binding variable regions of the humanized antibodies) willincrease blood clotting time by at least about 5 seconds when present inthe standard, assay at a concentration of at least about 1 nM to about20 nM, preferably about 5 nM to about 15 nM, more preferably about 10 nmin the assay. A typical control is a standard PT assay performed withoutadding any antibody of fragment. Additionally preferred antibodies andfragments of the invention achieve at least about 90% inhibition ofTF-dependent coagulation, preferably at least about 95% inhibition orgreater when compared to the control. A specific example of the standardPT assay is described in Examples 5 and 11.

Although a range of therapeutic anti-coagulant compositions of theinvention have been described above, other compositions that include thehumanized antibodies and fragments thereof are contemplated. Forexample, such antibodies and fragments may be used as the soletherapeutic agent or in combination with one or more other humanizedantibodies or fragments to achieve a desired outcome. Such antibodiesand fragments may also be used in combination with other antibodies,particularly human monoclonal antibodies reactive with other markers oncells responsible for the disease.

A wide spectrum of important uses for the present antibodies andfragments have been described above e.g., use to detect native TF in abiological sample, use to detect and purify cells expressing TF, and useto prevent or treat medical conditions such as undesired bloodcoagulation in a human patient. In practice, the humanized antibodiescan be used as separately administered compositions given in conjunctionwith other anti-clotting agents including aspirin, coumadin, heparin,hirudin, or hirulog. Also envisioned is co-administration withanti-platelet (e.g., ReoPro, Integrilin, Aggrestat, Plavix, and/orTiclid) and/or thrombolytic agents (e.g., tissue plasminogen activator,strepokinase and urokinase).

In embodiments in which the therapeutic anti-coagulant compositionsdescribed herein include one or more humanized antibodies or fragments,that composition may include a solution of the antibody or a cocktailthereof dissolved in an acceptable carrier, preferably an aqueouscarrier. A variety of aqueous carriers have already been referenced suchas water, buffered water, 0.4% saline, 0.3% glycine and the like. Thesesolutions are preferably sterile and generally free of particulatematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicityadjustment agents and the like, for example sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of antibody in these formulations can vary widely, forexample from less than about 0.5%, usually at or at least about 1% to asmuch as 15 or 20% by weight and will be selected primarily based onfluid volumes, viscosities, etc., in accordance with the particular modeof administration selected. See generally, Remington's PharmaceuticalSciences, supra.

If desired, the therapeutic anti-coagulant compositions described hereincan be lyophilized for storage and reconstituted in a suitable carrierprior to use. This technique has been shown to be effective withconventional immune globulins. Any suitable lyophilization andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilization and reconstitution can leadto varying degrees of antibody activity loss (e.g., with conventionalimmune globulins, IgM antibodies tend to have greater activity loss thanIgG antibodies) and that use levels may have to be adjusted tocompensate.

For some prophylactic applications, it will be helpful to administer thetherapeutic anti-coagulant compositions to a patient not already in adetectable disease state to enhance the patient's resistance to thedisease. Such an amount is defined to be a “prophylactically effectivedose”. In this use, the precise amounts again depend upon the patient'sstate of health and general level of immunity, but generally range from0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per patient. A preferredprophylactic use is for the prevention of undesired blood clottingfollowing a planned invasive medical procedure.

As discussed, the invention also features kits that include subjectantibodies or fragments thereof. In one embodiment, the humanizedantibodies or fragments thereof can be supplied for use against or inthe detection of TF antigen. Thus, for instance, one or more humanizedantibodies, fragments thereof, or single chain antibodies may beprovided, usually in a lyophilized form in a container. Such antibodies,fragments, or single chain antibodies, which may be conjugated to apreviously mentioned label or toxin, or unconjugated, are included inthe kits with buffers, such as Tris, phosphate, carbonate, etc.,stabilizers, biocides, inert proteins, e.g., serum albumin, or the like.Generally, these materials will be present in less than about 5% byweight based on the amount of active antibody, and usually present intotal amount of at least about 0.001% wt. based again on the antibodyconcentration. Frequently, it will be desirable to include an inertextender or excipient to dilute the active ingredients, where theexcipient may be present in from about 1 to 99% wt. of the totalcomposition. Where a second antibody capable of binding to the chimericantibody is employed in an assay, this will usually be present in aseparate vial. The second antibody is typically conjugated to a labeland formulated in an analogous manner with the antibody formulationsdescribed above. The kit will generally also include a set ofinstructions for use.

As discussed, the invention also provides a variety of methods ofinhibiting blood coagulation in a mammal, preferably a primate such as ahuman patient.

For example, in one embodiment, the methods include administering to themammal a therapeutically effective amount of at least one of, preferablyone, two or three of the humanized antibodies provided herein or afragment thereof that binds specifically to human tissue factor (TF) toform a complex. Typically, factor X or factor IX binding to TF orTF:FVIIa and activation by TF:FVIIa thereto is inhibited. In mostembodiments, the methods further include forming a specific complexbetween the antibody and the TF to inhibit the blood coagulation.

Also provided are methods of inhibiting blood coagulation in a mammalthat include administering to the mammal, a therapeutically effectiveamount of the humanized antibodies disclosed herein or a fragmentthereof. Typical antibodies and fragments bind specifically to humantissue factor (TF) to form a complex, and further wherein factor X orfactor IX binding to TF or TF:FVIIa and activation by TF:FVIIa theretois inhibited. In most embodiments, the methods further include forming aspecific complex between the antibody and the TF to inhibit the bloodcoagulation.

In a more specific example, the invention provides methods of inhibitingblood coagulation in a mammal that include administering to the mammal,a therapeutically effective amount of a humanized antibody or fragmentthereof disclosed herein. Typically, the antibody binds specifically tohuman tissue factor (TF) to form a complex, and further wherein factor Xor factor IX binding to TF or TF:FVIIa and activation by TF:FVIIathereto is inhibited. Preferably, the humanized antibody or fragmentincludes, on the heavy chain, at least one of and preferably all of thefollowing components:

a) a first CDR (CDR1) which is at least 95% identical to CDR1 amino acidsequence shown in FIG. 13B (SEQ ID NO: 8),

b) a second CDR (CDR2) which is at least 95% identical to the CDR2 aminoacid sequence shown in FIG. 13C (SEQ ID NOS: 9 or 101),

c) a third CDR (CDR3) which is at least 95% identical to the CDR3 aminoacid sequence shown in FIG. 13D (SEQ ID NO: 10),

d) a first framework (FR 1) which is at least 95% identical to the FR1amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO: 91) as“FR1 HC-08”,

e) a second framework (FR2) which is at least 95% identical to the FR2amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO: 91) as“FR2 HC-08”,

f) a third framework (FR3) which is at least 95% identical to the FR3amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO: 91) as“FR3 HC-08”,

g) a fourth framework (FR4) which is at least 95% identical to the FR4amino acid sequence shown in FIG. 13A (fragment of SEQ ID NO: 91) as“FR4 HC-08”.

In a more specific invention embodiment, the humanized antibodyincludes, on the light chain, at least one of, and preferably all of thefollowing components:

h) a first CDR (CDR1) which is at least 95% identical to CDR1 amino acidsequence shown in FIG. 12B (fragment of SEQ ID NO: 2),

i) a second CDR (CDR2) which is at least 95% identical to the CDR2 aminoacid sequence shown in FIG. 12C (SEQ ID NO: 6),

j) a third CDR (CDR3) which is at least 95% identical to the CDR3 aminoacid sequence shown in FIG. 12D (SEQ ID NO: 7),

k) a first framework (FR1) which is at least 95% identical to the FR1amino acid sequence shown in FIG. 12A (fragment of SEQ ID NO: 79) as“FRi LC-09”,

l) a second framework (FR2) which is at least 95% identical to the FR2amino acid sequence shown in FIG. 12A (fragment of SEQ ID NO: 79) as“FR2 LC-09”,

m) a third framework (FR3) which is at least 95% identical to the FR3amino acid sequence shown in FIG. 12A (fragment of SEQ ID NO: 79) as“FR3 LC-09”,

n) a fourth framework (FR4) which is at least 95% identical to the FR4amino acid sequence shown in FIG. 12A (fragment of SEQ ID NO: 79) as“FR4 LC-09”,

o) a light chain constant region which is at least 95% identical to theamino acid sequence shown in FIG. 14A (SEQ ID NO: 97) or FIG. 15A (SEQID NO: 99); and

p) a heavy chain constant region which is at least 95% identical to theamino acid sequence shown in FIG. 14B (SEQ ID NO: 98) or FIG. 15B (SEQID NO: 100).

In a more specific embodiment of the foregoing method, the humanizedantibody or fragment thereof includes, on the heavy chain, at least oneof and preferably all of the following components:

a) a first CDR (CDR1) identical to CDR1 amino acid sequence shown inFIG. 13B (SEQ ID NO: 8),

b) a second CDR (CDR2) identical to the CDR2 amino acid sequence shownin FIG. 13C (SEQ ID NOS: 9 or 101),

c) a third CDR (CDR3) identical to the CDR3 amino acid sequence shown inFIG. 13D (SEQ ID NO: 10),

d) a first framework (FR1) identical to the FR1 amino acid sequenceshown in FIG. 13A (fragment of SEQ ID NO: 91) as “FR1 HC-08”,

e) a second framework (FR2) identical to the FR2 amino acid sequenceshown in FIG. 13A (fragment of SEQ ID NO: 91) as “FR2 HC-08”,

f) a third framework (FR3) identical to the FR3 amino acid sequenceshown in FIG. 13A (fragment of SEQ ID NO: 91) as “FR3 HC-08”,

g) a fourth framework (FR4) identical to the FR4 amino acid sequenceshown in FIG. 13A (fragment of SEQ ID NO: 91) as “FR HC-08”;

and on the light chain:

h) a first CDR (CDR1) identical to CDR1 amino acid sequence shown inFIG. 12B (fragment of SEQ ID NO: 2),

i) a second CDR (CDR2) identical to the CDR2 amino acid sequence shownin FIG. 12C (SEQ ID NO: 6),

j) a third CDR (CDR3) identical to the CDR3 amino acid sequence shown inFIG. 12D (SEQ ID NO: 7),

k) a first framework (FR1) identical to the FR1 amino acid sequenceshown in FIG. 12A (fragment SEQ ID NO: 79) as “FRi LC-09”,

l) a second framework (FR2) identical to the FR2 amino acid sequenceshown in FIG. 12A (fragment SEQ ID NO: 79) as “FR2 LC-09”,

m) a third framework (FR3) identical to the FR3 amino acid sequenceshown in FIG. 12A (fragment SEQ ID NO: 79) as “FR3 LC-09”,

n) a fourth framework (FR4) identical to the FR4 amino acid sequenceshown in FIG. 12A (fragment SEQ ID NO: 79) as “FR4 LC-09”,

o) a light chain constant region which is identical to the amino acidsequence shown in FIG. 14A (SEQ ID NO: 79) or FIG. 15A (SEQ ID NO: 99),and

p) a heavy chain constant region which is identical to the amino acidsequence shown in FIG. 14B (SEQ ID NO: 98) or FIG. 15B (SEQ ID NO: 100).

The invention also provides for a variety of methods of detecting tissuefactor (TF) in a biological sample. In one embodiment, the methodincludes contacting a biological sample with the humanized antibodies orfragments thereof disclosed herein under conditions conducive to forminga complex and detecting the complex as being indicative of the TF in thebiological sample.

All documents mentioned herein are fully incorporated by reference intheir entirety.

EXEMPLIFICATION

The following non-limiting examples are illustrative of the invention.In the following examples and elsewhere the antibodies H36 and H36.D2are referred to. Those antibodies are the same antibody as H36.D2.B7,but H36 is derived from the mother clone, and H36.D2 is obtained fromthe primary clone, whereas H36.D2.B7 is obtained from the secondaryclone. No differences have been observed between those three clones withrespect to ability to inhibit TF or other physical properties. Ingeneral usage, H36 is often used to indicate anti-TF antibody producedby any of these clones or related cell lines producing the antibody.

EXAMPLE 1 Preparation and Cloning of Anti-rhTF Monoclonal Antibodies

Monoclonal antibodies against rhTF were prepared as follows.

A. Immunization and Boosts

Five female BALB/c mice were immunized with 10 μg each of lipidated,purified rhTF. The mice were initially sensitized intraperitoneallyusing Hunter's Titermax adjuvant. Three final boosts were administeredin 0.85% NaCi. Boosts were 2, 5.5, and 6.5 months post initialsensitization. All boosts were given intraperitoneally, except the firstwhich was subcutaneous. The final boost was given 3 days pre-fusion and20 μg was administered.

B. Fusion of Mouse Spleen Lymphocytes with Mouse Myeloma Cells

Lymphocytes from the spleen of one rhTF immunized BALB/c mouse was fusedto X63-Ag8.653 mouse myeloma cells using PEG 1500. Following exposure tothe PEG, the cells were incubated for one hour in heat inactivated fetalbovine serum at 37° C. The fused cells were then resuspended in RPMI1640 and incubated overnight at 37° C. with 10% CO₂. The cells wereplated the next day using RPMI 1640 and supplemented with macrophageculture supernatant.

C. ELISA Development

Plates for the ELISA assay were coated with 100 microliters ofrecombinant tissue factor (0.25 μg/ml) in a carbonate-based buffer. Allsteps were performed at room temperature. Plates were blocked with BSA,washed, and then the test samples and controls were added.Antigen/antibody binding was detected by incubating the plate with goatanti-mouse HRP conjugate (Jackson ImmunoResearch Laboratories) and thenusing an ABTS peroxidase substrate system (Kirkegaard and PerryLaboratories). Absorbance was read on an automatic plate reader at awavelength of 405 nm.

D. Stabilization of rhTF Hybridoma Cell Lines

Two weeks after fusion, screening of hybridoma colonies by specific rhTFELISA was started. Screening for new colonies continued for three weeks.The positive clones were tested every one to two weeks for continuedantibody production until fifteen stable clones were frozen down.

E. Primary and Secondary Cloning

Limiting dilution cloning was performed on each of the positive stablehybridomas to obtain primary clones. The cells were thawed, grown inculture for a short period of time, and then diluted from 10 cells/wellto 0.1 cells/well. Primary clones were tested by anti-rhTF ELISA andfive to six positive clones were expanded and frozen.

Secondary clone of anti-rhTF antibody, H36.D2.B7, was obtained fromprimary clone, H36.D2, prepared and stored in liquid nitrogen asdescribed above. Four different dilutions, 5 cells/well, 2 cells/well, 1cell/well, 0.5 cells/well of the primary clone were prepared in 96-wellsmicrotiter plates to start the secondary cloning. Cells were diluted inIMDM tissue culture media containing the following additives: 20% fetalbovine serum (FBS), 2 mM L-glutamine, 100 units/ml of penicillin, 100μg/ml of streptomycin, 1% GMS-S, 0.075% NaHCO₃. To determine clones thatsecrete anti-rhTF antibody, supernatants from five individual wells ofthe 0.2 cells/well microtiter plate were withdrawn after two weeks ofgrowth and tested for the presence of anti-rhTF antibody by ELISA assaysas described above. All five clones showed positive results in the ELISAassay, with H36.D2.B7 being the best antibody producer. All five cloneswere adapted and expanded in RPMI media containing the followingadditive: 10% FBS, 2 mM L-glutamine, 100 units/ml of penicillin, 100μg/ml of streptomycin, 1% GMS-S, 0.075% NaHCO₃, and 0.013 mg/ml ofoxalaacetic acid. H36.D2.B7 was purified by Protein A affinitychromatography from the supernatant of cell culture and was tested forits ability to inhibit TF:VIIa in a FX activation assay. The resultsindicated that H36.D2.B7 had the same inhibition as H36.D2 antibody. Allcells were stored in liquid nitrogen.

F. Isolation of total RNA from H36.D2.B7

269 μg of total RNA was isolated from 2.7×10⁵ H36.D2.B7 hybridoma cells.The isolation of total RNA was performed as described in the RNeasy MidiKits protocol from Qiagen. The RNA sample was stored in water at −20° C.until needed.

G. cDNA Synthesis and Cloning of Variable Regions of H36.D2.B7 Gene

To obtain the first strand of cDNA, a reaction mixture containing 5 μgof total RNA isolated as above, back primers JS300 (all primers areidentified below) for the heavy chain (HC) and OKA 57 for the lightchain (LC), RNase inhibitor, dNTP's, DTT, and superscript II reversetranscriptase, was prepared and incubated at 42° C. for 1 hour. Thereaction tube is then incubated at 65° C. for 15 minutes to stop thetranscription. After cooling down, five units of RNase H were then addedand the reaction was allowed to incubate at 37° C. for 20 minutes. ThecDNA sample was stored at −70° C. until needed.

PCR (polymerase chain reaction) was conducted separately to clone thevariable regions of both HC and LC of anti-rhTF, H36.D2.B7 from the cDNAmade as above (nucleic acid and amino acid sequences of those HC and LCvariable regions set forth in FIGS. 1A and 1B). Three rounds of PCR wereconducted. Round 1: PCR was run for 35 cycles at 96° C., 53° C. and 72°C. using front primer JS002 and back primer JS300 for HC. For LC frontprimer JS009 and back primer OKA 57 were used and PCR was rune for 35cycles at 96° C., 63° C. and 72° C. Round 2: PCR of both HC and LC wasrune the same as in Round 1 with the exception that pMC-18 was used forHC front primer and pMC-15 for LC front primer. Round 3: PCR was run for30 cycles at 96° C., 60-65° C. and 72° C. using H36HCF and H36HCRprimers HC. For LC, PCR was run for 30 cycles at 96° C., 58° C. and 72°C. using H36LCF and H36LCR primers.

The following primers were used for cloning H36.D2.B7 variable regionsof HC and LC. OKA 57: 5′-GCACCTCCAGATGTTAACTGCTC-3′ (SEQ ID NO: 17)J5300: 5′-GAARTAVCCCTTGACCAGGC-3′ (SEQ ID NO: 18) JS009:5′-GGAGGCGGCGGTTCTGACATTGTGMTGWCMC (SEQ ID NO: 19) ARTC-3′ JS002:5′-ATTTCAGGCCCAGCCGGCCATGGCCGARGTY (SEQ ID NO: 20) CARCTKCARCARYC-3′pMC-15: 5′-CCCGGGCCACCATGKCCCCWRCTCAGYTYCT (SEQ ID NO: 21) KG-3′ pMC-18: 5′-CCCGGGCCACCATGGRATGSAGCTGKGTMAT (SEQ ID NO: 22) SCTC-3′ H36HCF:5′-ATATACTCGCGACAGCTACAGGTGTCCACTC (SEQ ID NO: 23)CGAGATCCAGCTGCAGCAGTC-3′ H36HCR: 5′-GACCTGAATTCTAAGGAGACTGTGAGAGTG (SEQID NO: 24) G-3′ H36LCF: 5′-TTAATTGATATCCAGATGACCCAGTCTC (SEQ ID NO: 25)C-3′ H36LCR: TAATCGTTCGAAAAGTGTACTTACGTTTCAGCTC (SEQ ID NO: 26)CAGCTTGGTCCwherein in the above SEQ ID NOS: 17 through 26: K is G or T; M is A orC; R is A or G; S is C or G; V is A, C or G; W is A or T; Y is C or T.

EXAMPLE 2 Binding Activity of Antibodies of the Invention

Antibodies of the invention as prepared in Example 1 above wereemployed. The rhTF molecule was expressed in E. coli and purified byimmunoaffinity chromatography in accordance with standard methods (seeHarlow and Lane, supra, Ausubel et al. supra). Antibody association(K_(a)) and dissociation (K_(d)) constants were determined by ELISA andsurface plasmon resonance (i.e., BIACore) assays (see e.g., Harlow andLane, supra; Ausubel et al. supra; Altschuh et al., Biochem., 31:6298(1992); and the BIAcore method disclosed by Pharmacia Biosensor). ForBIACore assays, rhTF was immobilized on a biosensor chip in accordancewith the manufacturer's instructions. Constants for each antibody weredetermined at four antibody concentrations (0.125 nM, 0.25 nM, 0.5 nM,and 1 nM).

Protein concentrations were determined by standard assay (M. M.Bradford, Anal. Biochem., 72:248 (1976)) using Bovine Serum Albumin as astandard and a commercially available dye reagent (Bio-Rad).

FIG. 2 shows association and disassociation constants for each anti-TFantibody. Antibody H36 exhibited the highest association rate(K_(a)=3.1×10¹⁰ M⁻¹) and the lowest disassociation rate (K_(d)=3.2×10⁻¹¹M) of any of the anti-TF antibodies tested.

EXAMPLE 3 FXa-Specific Substrate Assay

In general, the experiments described herein were conducted using rhTFlipidated with phosphatidycholine (0.07 mg/ml) and phosphatidylserine(0.03 mg/ml) at a 70/30 w/w ratio in 50 mM Tris-HCl, pH 7.5, 0.1% bovineserum albumin (BSA) for 30 minutes at 37° C. A stock solution ofpreformed TF:FVIIa complex was made by incubating 5 nM of the lipidatedrhTF and 5 nM of FVIIa for 30 minutes at 37° C. The TF:FVIIa complex wasaliquoted and stored at −70° C. until needed. Purified human factorsVII, VIIa, and FX were obtained from Enyzme Research Laboratories, Inc.The following buffer was used for all FXa and FVIIa assays: 25 mMHepes-NaOH, 5 mM CaCl₂, 150 mM NaCl, 0.1% BSA, pH 7.5.

Monoclonal antibodies were screened for capacity to blockTF:VIIa-mediated activation of FX to FXa. The FX activation wasdetermined in two discontinuous steps. In the first step (FXactivation), FX conversion to FXa was assayed in the presence of Ca⁺².In the second step (FXa activity assay), FX activation was quenched byEDTA and the formation of FXa was determined using a FXa-specificchromogenic substrate (S-2222). The S-2222 and S-2288 (see below)chromogens were obtained from Chromogenix (distributed by PharmaciaHepar Inc.). FX activation was conducted in 1.5 ml microfuge tubes byincubating the reaction with 0.08 nM TF:VIIa, either pre-incubated withan anti-rhTF antibody or a buffer control. The reaction was subsequentlyincubated for 30 minutes at 37° C., then 30 nM FX was added followed byan additional incubation for 10 minutes at 37° C. FXa activity wasdetermined in 96-well microtiter plates. Twenty microliters of samplewas withdrawn from step one and admixed with an equal volume of EDTA(500 mM) in each well, followed by addition of 0.144 ml of buffer and0.016 ml of 5 mM S-2222 substrate. The reaction was allowed to incubatefor an additional 15-30 minutes at 37° C. Reactions were then quenchedwith 0.05 ml of 50% acetic acid, after which, absorbance at 405 nm wasrecorded for each reaction. The inhibition of TF:FVIIa activity wascalculated from OD_(405nm) values in the experimental (plus antibody)and control (no antibody) samples. In some experiments, an anti-hTFantibody, TF:FVIIa, and FX were each added simultaneously to detectbinding competition. FIG. 3 shows that the H36.D2 MAb (in bold)inhibited TF:FVIIa activity toward FX to a significantly greater extent(95%) than other anti-rHTF Mabs tested.

EXAMPLE 4 FVIIa-Specific Substrate Assay

Monoclonal antibodies were further screened by an FVIIa specific assay.In this assay, 5 nM lipidated rhTF was first incubated with buffer(control) or 50 nM antibody (experimental) in a 96-well microtiter platefor 30 minutes at 37° C., then admixed with 5 nM purified human FVIIa(V_(T)=0.192 ml), followed by 30 minutes incubation at 37° C. Eightmicroliters of a 20 mM stock solution of the FVIIa specific substrateS-2288 was then added to each well (final concentration, 0.8 mM).Subsequently, the reaction was incubated for one hour at 37° C.Absorbance at 405 nm was then measured after quenching with 0.06 ml of50% acetic acid. Percent inhibition of TF:FVIIa activity was calculatedfrom OD_(405nm) values from the experimental and control samples.

FIG. 4 shows the H36 antibody did not significantly block TF:FVIIaactivity toward the S-2288 substrate when the antibody was eitherpre-incubated with TF (prior to FVIIa addition) or added to TFpre-incubated with FVIIa (prior to adding the antibody). This indicatesthat H36 does not interfere with the interaction (binding) between TFand FVIIa, and that H36 also does not inhibit TF:FVIIa activity toward apeptide substrate.

EXAMPLE 5 Prothrombin Time (PT) Assay

Calcified blood plasma will clot within a few seconds after addition ofthromplastin (TF); a phenomenon called the “prothrombin time” (PT). Aprolonged PT is typically a useful indicator of anti-coagulationactivity (see e.g., Gilman et al. supra).

The H36.D2 antibody was investigated for capacity to affect PT accordingto standard methods using commercially available human plasma (Ci-TrolControl, Level I obtained from Baxter Diagnostics Inc.). Clot reactionswere initiated by addition of lipidated rhTF in the presence of Ca⁺².Clot time was monitored by an automated coagulation timer (MLA Electra800). PT assays were initiated by injecting 0.2 ml of lipidated rhTF (ina buffer of 50 mM Tris-HCl, pH 7.5, containing 0.1% BSA, 14.6 mM CaCl₂,0.07 mg/ml of phosphatidylcholine, and 0.03 mg/ml of phosphatidylserine)into plastic twin-well cuvettes. The cuvettes each contained 0.1 ml ofthe plasma preincubated with either 0.01 ml of buffer (control sample)or antibody (experimental sample) for 1-2 minutes. The inhibition ofTF-mediated coagulation by the H36.D2 antibody was calculated using a TFstandard curve in which the log [TF] was plotted against log clot time.

FIG. 5 shows the H36.D2 antibody substantially inhibits TF-initiatedcoagulation in human plasma. The H36.D2 antibody increased PT timessignificantly, showing that the antibody is an effective inhibitor ofTF-initiated coagulation (up to approximately 99% inhibition).

EXAMPLE 6 FX and H36.D2 Antibody Compete for Binding to the TF:FVIIaComplex

Competition experiments were conducted between TF:FVIIa, FX and theH36.D2 antibody. FIG. 6A illustrates the results of an experiment inwhich a preformed TF:FVIIa complex (0.08 nM) was pre-incubated at 37° C.for 30 minutes in buffer including 0.02 nM, 0.04 nM, 0.08 nM and 0.16 nMof the H36.D2 monoclonal antibody, respectively. FX (30 nM) was thenadded to the TF:FVIIa and H36.D2 antibody mixture and the mixtureallowed to incubate for an additional 10 minutes at 37° C. FX activationwas quenched with EDTA as described previously. The FXa produced therebywas determined by the FXa-specific assay described in Example 3, above.

FIG. 6B shows the results of an experiment conducted along the linesjust-described, except that the H36.D2 antibody, pre-formed TF:FVIIa,and FX were added simultaneously to start the FX activation assay.

The data set forth in FIGS. 6A and 6B show that the H36.D2 antibody andFX compete for binding to the pre-formed TF:FVIIa complex.

EXAMPLE 7 Inhibition of TF Activity in Cell Culture

J-82 is a human bladder carcinoma cell line (available from the ATCC)which abundantly expresses native human TF as a cell surface protein. Tosee if the H36.D2 antibody could prevent FX from binding to native TFdisplayed on the cell surface, a J-82 FX activation assay was conductedin microtiter plates in the presence of FVII (see D. S. Fair et al., J.Biol. Chem., 262:11692 (1987)). To each well, 2×10⁵ cells was added andincubated with either 50 ng FVII, buffer (control sample) or the anti-TFantibody (experimental sample) for 2 hours at 37° C. Afterwards, eachwell was gently washed with buffer and 0.3 ml of FX (0.05 mg/ml) wasadded to each well for 30 minutes at room temperature. In some cases,the antibody was added at the same time as FX to detect bindingcompetition for the native TF. Thereafter, 0.05 ml aliquots were removedand added to new wells in a 96-well microtiter plate containing 0.025 mlof 100 mM EDTA. FXa activity was determined by the FXa-specific assay asdescribed in Example 3, above. Inhibition of TF activity on the surfaceof the J-82 cells was calculated from the OD_(405nm) in the absence(control sample) and presence of antibody (experimental sample).

FIG. 7 shows that the H36.D2 antibody bound native TF expressed on J-82cell membranes and inhibited TF-mediated activation of FX. These resultsindicate that the antibody competes with FX for binding to native TFdisplayed on the cell surface. Taken with the data of Example 8, below,the results also show that the H36.D2 antibody can bind a conformationalepitope on native TF in a cell membrane.

EXAMPLE 8 Specific Binding of the H36.D2 Antibody to Native rhTF

Evaluation of H36.D2 binding to native and non-native rhTF was performedby a simplified dot blot assay. Specifically, rhTF was diluted to 30μg/ml in each of the following three buffers: 10 mM Tris-HCl, pH 8.0; 10mM Tris-HCl, pH 8.0 and 8 M urea; and 10 mM Tris-HCl, pH 8.0, 8 M ureaand 5 mM dithiothreitol. Incubation in the Tris buffer maintains rhTF innative form, whereas treatment with 8M urea and 5nM dithiothreitolproduces non-native (denatured) rhTF. Each sample was incubated for 24hours at room temperature. After the incubation, a Millipore Immobilon(7×7 cm section) membrane was pre-wetted with methanol, followed by 25mM Tris, pH 10.4, including 20% methanol. After the membranes wereair-dried, approximately 0.5 μl, 1 μl, and 2 μl of each sample (30μg/ml) was applied to the membrane and air-dried. After blocking themembrane by PBS containing 5% (w/v) skim milk and 5% (v/v) NP-40, themembrane was probed with H36.D2 antibody, followed by incubation with agoat anti-mouse IgG peroxidase conjugate (obtained from JacksonImmunoResearch Laboratories, Inc.). After incubation with ECL WesternBlotting reagents in accordance with the manufacturer's instructions(Amersham), the membrane was wrapped with plastic film (Saran Wrap) andexposed to X-ray film for various times.

FIG. 8A shows that the H36.D2 monoclonal antibody binds a conformationalepitope on native TF in the presence of Tris buffer or Tris buffer with8M urea (lanes 1 and 2). The autoradiogram was exposed for 40 seconds.However, when the native TF was denatured with 8M urea and 5mM DTT,H36.D2 binding was significantly reduced or eliminated (lane 3). FIG. 8Bshows an over-exposed autoradiogram showing residual binding of theH36.D2 antibody to non-native (i.e., denatured) rhTF. The over-exposurewas for approximately 120 seconds. Treatment with 8M urea alone probablyresulted in only partial denaturation of the native rhTF since the twodisulfide bonds in TF are not reduced. It is also possible that thepartially denatured TF may refold back to native confirmation duringlater blotting process when urea is removed. These results also clearlydistinguish preferred antibodies of the invention which do not binddenatured TF from previously reported antibodies which do notselectively bind to a conformational epitope and bind to denatured TF(see U.S. Pat. No. 5,437,864 where in FIG. 18 Western Blot analysisshows binding to TF denatured by SDS).

EXAMPLE 9 Humanization of Anti-Tissue Factor Antibody

The previous examples describe how to make and use a particular murineantibody called H36.D2 (sometimes also called H36 as discussed above).The present example shows how to make and use a humanized version ofthat antibody. A humanized H36 antibody has a variety of uses includinghelping to minimize potential for human anti-mouse antibody (HAMA)immunological responses. These and other undesired responses poseproblems for use of the H36 antibody in human therapeutic applications.

A. Preparation of Chimeric Anti-Tissue Factor Antibody (cH36)

The H36 antibody described previously is an IgG2a murine antibody. H36was first converted to a mouse-human chimeric antibody for clinicaldevelopment. To do this, the heavy and light chain genes for H36 werecloned (see U.S. Pat. No.5,986,065). The heavy chain variable region wasfused to a human IgG4 constant (Fc) domain and the light chain variableregion was fused to a human kappa light chain constant domain. Theresulting IgG4κ chimeric antibody was designated Sunol-cH36. Formultiple uses of H36 or cH36 in patients with chronic diseases, a fullyhumanized cH36 is preferred so that it will decease or eliminate anyhuman anti-mouse antibody immunological response. The humanization ofcH36 is described below.

B. Humanization of cH36 Antibody Humanization of the chimericanti-tissue factor antibody cH36 was achieved by using a “best-fit”method. This method takes full advantage of the fact that a great numberof human IgGs with known amino acid sequences are available in thepublic database. The individual frameworks of the mouse heavy and lightvariable regions in cH36 are compared with their corresponding humanframeworks in the Kabat database (see http://immuno.bme.nwu.edu). Thefollowing criteria were used to select the desired human IgG frameworksfor humanization: (1) The number of mismatched amino acids was kept aslow as possible. (2) Amino acids inside the “vernier” zone (amino acidsin this zone may adjust CDR structure and fine-tune the fit to antigen,see Foote, J. and Winter, G., J. of Mol. Bio. 224, (2) 487-499 [1992])were left unchanged. (3) Conservative amino acid substitutions werefavored when evaluating similar candidates. The matching program usedfor this comparison can be found in Kabat's home page atimmuno.bme.nwu.edu (Johnson G, Wu T. “Kabat database and itsapplication: Future directions.” Nucleic Acids Res. (2001) 29:205-206).The program finds and aligns regions of homologies between the mousesequences and human sequences in the Kabat's database. By using thisunique best-fit method, it is anticipated that the humanized LC or HCvariable region of the target IgG may have all the four FRs derived fromas few as one human IgG molecule or to as many as four different humanIgG molecules.

(i). Selection of Human IgG Kappa Light Chain Variable Region Frameworks

The amino acid sequence in each of the frameworks of cH36 LC wascompared with the amino acid sequence in the corresponding FR in humanIgG kappa light chain variable region in Kabat Database. The best-fit FRwas selected based on the three creteria described above.

The amino acid sequence of human IgG kappa light chain variable regionwith a Kabat Database ID No. 005191 was selected for humanization ofcH36 LC FR1. The amino acid sequence of human IgG kappa light chainvariable region with a Kabat Database ID No. 019308 was selected forhumanization of cH36 LC FR2. The following mutations were made in cH36LC FR1 to match the amino acid sequence of a human IgG kappa light chainvariable region with a Kabat Database ID No. 005191: Q11→L, L15→V,E17→D, S18→R. One mutation Q37→L was made cH36 LC FR2 to match the aminoacid sequence of a human IgG kappa light chain variable region with aKabat Database ID No. 019308 (see Table 1A for sequence information).

The amino acid sequence of a human IgG kappa light chain variable regionwith a Kabat Database ID No. 038233 was selected for humanization ofcH36 LC FR3. The amino acid sequence of a human IgG kappa light chainvariable region with a Kabat Database ID No. 004733 was selected forhumanization of cH36 LC FR4. The following mutations were made in cH36LC FR3 to match the amino acid sequence of a human IgG kappa light chainvariable region with a Kabat Database ID No. 038233: K70→D, K74→T,A80→P, V84→A, N85→T. Two mutations A100→Q and L106→I were made cH36 LCFR4 to match the amino acid sequence of a human IgG kappa light chainvariable region with a Kabat Database ID No. 004733 (see Table 1B forsequence information).

(ii). Selection of Human IgG Heavy Chain Variable Region Frameworks

The amino acid sequence in each of the frameworks of cH36 HC wascompared with the amino acid sequence in the corresponding FR in humanIgG heavy chain variable region in Kabat Database. The best-fit FR wasselected based on the three criteria described above.

The amino acid sequence of a human IgG heavy chain variable region witha Kabat Database ID No. 000042 was selected for humanization of cH36 HCFR1. The amino acid sequence of a human IgG heavy chain variable regionwith a Kabat Database ID No. 023960 was selected for humanization ofcH36 HC FR2. The following mutations were made in cH36 HC FR1 to matchthe amino acid sequence of a human IgG heavy chain variable region witha Kabat Database ID No.000042: E1→Q, Q5→V, P9→G, L11→V, V12→K, Q19→R,T24→A. Two mutations H41→P and S44→G were made cH36 HC FR2 to match theamino acid sequence of a human IgG heavy chain variable region with aKabat Database ID No. 023960 (see Table 2A for sequence information).

The amino acid sequence of a human IgG heavy chain variable region witha Kabat Database ID No. 037010 was selected for humanization of cH36 HCFR3. The amino acid sequence of a human IgG heavy chain variable regionwith a Kabat Database ID No. 000049 was selected for humanization ofcH36 HC FR4. The following mutations were made in cH36 HC FR3 to matchthe amino acid sequence of a human IgG heavy chain variable region witha Kabat Database ID No. 037010: S76→T, T77→S, F80→Y, H82→E, N84→S,T87→R, D89→E, S91→T. One mutations L113→V was made cH36 HC FR2 to matchthe amino acid sequence of a human IgG heavy chain variable region witha Kabat Database ID No. 000049 (see Table 2B for sequence information).

Table 1A and 1B: Comparison of cH36 and Human Light Chain (LC) FRSequences TABLE 1A FR1 (23 AA) FR2 (15 AA) Names 1        10       2035          48 (SEQ ID NO: 102) DIQMTQSPASQSASLGESVTITC WYQQKPGKSPQLLIYcH36-LC (SEQ ID NO: 27) DIQMTQSPASLSASVGDRVTITC WYLQKPGKSPQLLIY Human LC

TABLE 1B FR3 (32 AA) FR4 (10 AA) Names 57 60        70       80     8698       107 GVPSRFSGSGSGTKFSFKISSLQAEDFVNYYC  FGAGTKLELK cH36-LC(fragment of SEQ ID NO: 72) GVPSRFSGSGSGTDFSFTISSLQPEDFATYYC  FGQGTKLEIKHuman-LC (SEQ ID NO: 28)

Table 2A and 2B: Comparison of cH36 and Human Heavy Chain (HC) FRSequences TABLE 2A FR1 (30 AA) FR2 (14 AA) Names1        10       20        29 36       44EIQLQQSGPELVKPGASVQVSCKTSGYSFT  WVRQSHGKSLEWIG cH3G-HC (fragment of SEQID NO: 83) QIQLVQSGGEVKKPGASVRVSCKASGYSFT  WVRQSPGKGLEWIG Human-HC (SEQID NO:29)

TABLE 2B FR3 (32 AA) FR4 (11 AA) Names 67      75        85        95107       117 KATLTVDKSSTTAFMHLNSLTSDDSAVYFCAR  WGQGTTLTVSS cH3G-HC(fragment of SEQ ID NO:83) KATLTVDKSTSTAYMELSSLRSEDTAVYFCAR  WGQGTTVTVSSHuman-HC (SEQ ID NO:30)

Once the decisions on the desired human frameworks were made, thefollowing three techniques were used to achieve the desired amino acidsubstitutions in both the light and heavy chains: (1) Regular PCR wasused for cloning, to introduce cloning or diagnostic endonuclease sites,and to change amino acid residues located at the ends of the variableregions. (2) PCR-based mutagenesis was used to change multiple aminoacid residues at a time, especially when these residues were in themiddle of the variable regions. (3) Site directed mutagenesis was usedto introduce one or two amino acid substitutions at a time. Sitedirected mutagenesis was done following the protocol described inStratagene's “QuickChange Site-Directed Mutagenesis Kit” (Catalog#200518).

After each step, the partially humanized clones were sequenced and someof these variable regions were later cloned into expression vectors. Theplasmid tKMC180 was used to express LC mutants, and pJRS 355 or pLAM 356vector was used to express HC mutants as IgG1 or IgG4, respectively.Some of these clones were then combined and expressed transiently in COScells to determine the expression levels by ELISA.

The final fully humanized forms of the anti-TF heavy and light variableregions were cloned into what is sometimes referred to herein as a “megavector” and transfected into CHO and NSO cells for IgG expression.Stable cell lines were then used to produce amounts of humanized anti-TFsufficient for analysis. The resulting humanized versions are 100% humanin origin (when the CDR sequences are not considered). The humanizedIgG4 kappa version is designated hFAT (humanized IgG Four Anti-TissueFactor antibody) and the IgG1 kappa version is designated hOAT(humanized IgG One Anti-Tissue Factor antibody). These fully humanizedversions of cH36 are intended for treating chronic indications, such asthrombosis, cancer and inflammatory diseases.

C. Humanization of Anti-TF Antibody Heavy Chain

-   -   1. PCR amplification and cloning into pGem T-easy of anti-TF mAb        cH36 heavy chain (HC) variable region were performed using        plasmid pJAIgG4TF.A8 (an expression vector for chimeric H36) as        template and primers TFHC1s2 and TFHC1as2. Primer TFHC1s2        introduced a BsiW1 site upstream of the initiation codon and        also an amino acid change E1 to Q in framework (FR) 1. Primer        TFHC1as introduced an amino acid change L113 to V in FR4. This        step resulted in the construct HC01.    -   2. PCR-based mutagenesis using the previous construct (HC01) and        the following four primers generated construct HC02. Upstream        PCR used primers TFHC1s2 and TFHC7as. Downstream PCR used        primers TFHC7s and TFHC1as2. Overlap PCR using upstream and        downstream PCR products as templates and with primers TFHC1s2        and TFHC1as2 yielded HC02. The use of primers TFHC7s and TFHC7as        introduced two amino acid changes in FR2: H41 to P and S44 to G.    -   3. PCR-based mutagenesis using HC02 as template and the        following four primers generated construct HC03. Upstream PCR        used primers TFHC1s2 and TFHC5as2. Downstream PCR used primers        TFHC5s and TFHC1as2. PCR using upstream and downstream PCR        products as templates and with primers TFHC1s2 and TFHC1as2        yielded HC03. The use of primers TFHC5s and TFHC5as2 introduced        three amino acid changes in FR3: T87 to R, D89 to E, and S91        to T. A Bgl II site was also introduced at position. 87.    -   4. PCR amplification was performed using primers TFHC2s and        TFHC3as and HC03 in pGem as template. TFHC2s sits upstream of        the cloning site in pGem. TFHC3as sits in framework 3 and        introduces two amino acid changes in FR3: H82 to E and N84 to S.        The resulting PCR band was cloned into pGem and then the proper        size insert was digested with BsiW1 and Bgl H. Cloning of this        fragment into HC03 yields HC04.    -   5. PCR-based mutagenesis using HC04 as template and the        following primers resulted in HC05. Upstream PCR used primers        TFHC1s2 and TFHC6as. Downstream PCR used primers TFHC6s and        TFHC1as2. Mutagenic PCR using upstream and downstream PCR        products as templates and with primers TFHC1s2 and TFHC1as2        yielded HC05. This step introduced the following amino acid        changes in FR3: S76 to T, T77 to S, and F80 to Y.    -   6. PCR-based mutagenesis using HC05 as template and the        following four primers generated HC06. Upstream PCR used primers        TFHC2s and TFHC2as2. Downstream PCR used primers TFHC3s2 and        TFHC1as2. Amplification using TFHC2as2 introduced an amino acid        change in FR1: P9 to G. Primer TFHC3s2 changes Q19 to R and T24        to A. PCR using upstream and downstream PCR products as template        and with primers TFHC1 s2 and TFHC1 as2 yielded HC06.    -   7. A point mutation from I to M in position 2 of FR1 was        spontaneously introduced during construction of HC06. PCR        amplification using HC06 as template and TFHC1s3 and TFHC1as2 as        primers, corrected this erroneous substitution and also        introduced an amino acid. change in FR1: Q5 to V. The resulting        construct was HC07.    -   8. Construct HC08 was made by PCR-based mutagenesis using HC07        as template and the following primers. TFHC2s and TFHC2as3 were        used for the upstream product. The downstream product was        previously amplified using TFHC1s3 and TFHC1as2 (see step 7).        The use of primer TFHC2as3 introduced two amino acid changes in        FR1: L11 to V and V12 to K. A spontaneous point mutation        resulted in a F to L change at position 64 in CDR2. Further        screening and sequencing yielded construct HC08R1, which has the        correct sequence of F at position 64 in CDR2.    -   9. Two constructs, HC11 and HC12, were generated by        site-directed mutagenesis from HC07. Two complementary primers        TFHC8sP and TFHC8asP were used along with HC07 as template to        produce HC11 which contains three amino acid changes in FR1: G9        P, L11 to V, and V12 to K. Then, HC11 was methylated and column        purified for the next round of site directed mutagenesis. PCR        using HC11 as a template and the complementary primers TFHC9sL        and TFHC0asL generated HC12 which has a mutation from V11 to L        in FR1.    -   10. Construct HC09 was derived from HC12 by performing PCR using        HC12 as a template and the complementary primers TFHC10sK and        TFHC10asK. HC09 contains an amino acid change: K12 to V in FR1.    -   11. Construct HC10 was made from HC09. PCR using HC09 as a        template and the complementary primers LV-1 and LV-2 resulted in        the generation of HC10, which contains a mutation from L11 to V        in FR1.

After each mutation step, the partially humanized or fully humanizedclones were sequenced and some of these variable regions were latercloned into expression vectors. pJRS 355 or pLAM 356 vector was used toexpress HC mutants fused to Fc of human IgG1 or IgG4.

FIG. 13A summarizes steps 1-11 and shows incremental amino acid changesintroduced into FR1-4. Except HC08, all other heavy chain mutants andcH36 contain F at position 64 in CDR2. HC08 has a mutation from F to Lat position 64. FIGS. 13B-D show the heavy chain CDR sequences.

Primers Used for Heavy Chain Humanization TFHC1s25′ TTTCGTACGTCTTGTCCCAGATCCAGCTGCA (SEQ ID NO: 31) GCAGTC 3′ TFHC1as25′ AGCGAATTCTGAGGAGACTGTGACAGTGGTG (SEQ ID NO: 32) CCTTGGCCCCAG 3′TFHC7s 5′ GTGAGGCAGAGCCCTGGAAAGGGCCTTGAGT (SEQ ID NO: 33) GGATTGG 3′TFHC7as 5′ CCAATCCACTCAAGGCCCTTTCCAGGGCTCT (SEQ ID NO: 34) GCCTCAC 3′TFHC5s 5′GCATCTCAACAGCCTGAGATCTGAAGACACTGCAG (SEQ ID NO: 35)TTTATTTCTGTG 3′ TFHC5as2 5′ CTGCAGTGTCTTCAGATCTCAGGCTGTTGAG (SEQ ID NO:36) ATGCATGAAGGC 3′ TFHC3as 5′ GTCTTCAGATCTCAGGCTGCTGAGCTCCATG (SEQ IDNO: 37) AAGGCTGTGGTG 3′ TFHC2s 5′ TACGACTCACTATAGGGCGAATTGG 3′ (SEQ IDNO: 38) TFHC6s 5′ CTGTTGACAAGTCTACCAGCACAGCCTACAT (SEQ ID NO: 39)GGAGCTCAGCAG 3′ TFHC6as 5′ CTGCTGAGCTCCATGTAGGCTGTGCTGGTAG (SEQ ID NO:40) ACTTGTCAACAG 3′ TFHC2as2 5′ GCACTGAAGCCCCAGGCTTCACCAGCTCACC (SEQ IDNO: 41) TCCAGACTGCTGCAGC 3′ TFHC3s25′CTGGGGCTTCAGTGCGGGTATCCTGCAAGGCTTCT (SEQ ID NO: 42) GGTTACTCATTCAC 3′TFHC1s3 5′ TCGTACGTCTTGTCCCAGATCCAGCTGGTGC (SEQ ID NO: 43)AGTCTGGAGGTGAGC 3′ TFHC2as3 5′ GCACTGAAGCCCCAGGCTTCTTCACCTCACC (SEQ IDNO: 44) TCCAGACTGCACC 3′ TFHC9sL 5′ GCAGTCTGGACCTGAGCTGAAGAAGCCTGG (SEQID NO: 45) GG 3′ TFHC9asL 5′ CCCCAGGCTTCTTCAGCTCAGGTCCAGACT (SEQ ID NO:46) GC 3′ TFHC8sP 5′ GCTGGTGCAGTCTGGACCTGAGGTGAAGAAG (SEQ ID NO: 47) CC3′ TFHC8asP 5′ GGCTTCTTCACCTCAGGTCCAGACTGCACCA (SEQ ID NO: 48) GC3′TFHC10sK 5′ GCAGTCTGGACCTGAGCTGGTGAAGCCTGGG (SEQ ID NO: 49) GCTTC 3′TFHC10asK 5′ GAAGCCCCAGGCTTCACCAGCTCAGGTCCAG (SEQ ID NO: 50) ACTGC 3′LV-1 5′ CAGTCTGGACCTGAGGTGGTGAAGCCTG (SEQ ID NO: 51) GG 3′ LV-25′ CCCAGGCTTCACCACCTCAGGTCCAGA (SEQ ID NO: 52) CTG 3′

D. Humanization of Anti-TF Antibody Light Chain

-   -   1. PCR amplification was performed using plasmid pJAIgG4TF.A8        (an expression vector for chimeric H36) as template and primers        TFLC1s2.1 and TFLC1as2. This step introduced a cloning site,        AgeI, upstream of the coding region. It also introduced the        L1061 mutation in FR4. This step yielded the construct LC03.    -   2. Site-directed mutagenesis was performed using complementary        primers TFLC5s and TFLC5as and LC03 as template. This step        introduced the mutation Q37L in FR2 and added a PstI site for        diagnostic purposes. This new construct is named LC04.    -   3. PCR amplification was performed using LC04 as template and        primers TFHC2s and TFLC2as1. This step generated Fragment A that        will be used in step 6. This step introduced Q11L and L15V        mutations in FR1.    -   4. PCR amplification was performed using LC04 as template and        primers TFLC1s2.1 and TFLC1asR. This introduced the KpnI site at        the end of LC variable region. Cloning of this PCR fragment into        pGEM yields pGEM04K that will be used in step 6.    -   5. PCR amplification was performed using LC04 as template and        primers TFLC2s and TFLC4as. This step generated Fragment C that        will be used in step 6. Three mutations E17D, S18R in FR1 and        A100Q in FR4 were introduced in this step.    -   6. PCR-based mutagenesis using Fragment A and Fragment C as        templates and primers TFHC2s and TFLC4as yielded Fragment D.        Cloning of Fragment D into pGEM04K yielded the construct LC05.    -   7. PCR amplification was performed using pGEM04K as template and        primers TFLC1s2.1 and TFLC4as. This step generated Fragment H,        which is then cloned into pGEM04K. This introduced the A100Q        mutation in FR4 and the construct is named LC06.    -   8. PCR amplification was performed using LC06 as template and        primers TFLC1s2.1 and TFLC3as. This step generated Fragment I        that will be used in step 10. This introduced the K70D and the        K74T mutations in FR3.    -   9. PCR amplification was performed using LC06 as template and        primers TFLC3s2 and TFLC4as. This step generated Fragment F that        will be used in step 10. This introduced the A80P mutation in        FR3.    -   10. PCR using Fragment I and Fragment F as templates and primers        TFLC1 s2.1 and TFLC4as yielded Fragment J. Cloning of Fragment J        into pGEM yielded the construct LC07.    -   11. Site-directed mutagenesis was conduced using complementary        primers TFLC08sds and TFLC08sdsa and LC07 as template. This step        introduced the mutations V84A and N85T in FR3. This construct is        named LC08.    -   12. The AgeI to EcoO109I fragment from LC05 containing the        mutations Q11L, L15V, E17D, S18R and Q37L is cloned into LC08.        This yielded the construct LC09.    -   13. Site-directed mutagenesis was conduced using LC09 as        template and the complementary primers LC105 and LC103. This        step introduced the T85N mutation in FR3 and yielded the        construct LC10.    -   14. Site-directed mutagenesis was conducted using LC10 as        template and the complementary primers LC115 and LC113. This        step introduced the D70K mutation in FR3. This yielded the        construct LC11.    -   15. Site-directed mutagenesis was conducted using LC11 as        template and the complementary primers LC125a and LC123a. This        step introduced the K42Q mutation in FR2. This yielded the        construct LC12.

After each mutation step, the partially humanized or fully humanized LCclones were sequenced and some of these variable regions were latercloned into expression vector tKMC180.

FIG. 12 A summarizes steps 1-15 and shows incremental amino acid changesintroduced into FR1-4 of the light chain. FIGS. 12B-D show the lightchain CDR sequences.

Oligonucleotide Primers Used for Light Chain Humanization TFLC1as2:5′ TTCGAAAAGTGTACTTACGTTTGATCTCCAG (SEQ ID NO: 53) CTTGGTCCCAG 3′TFLC1s2.1: 5′ ACCGGTGATATCCAGATGACCCAGTCT (SEQ ID NO: 54) CC 3′ TFLC5s:5′ GGTTAGCATGGTATCTGCAGAAACCAG (SEQ ID NO: 55) GG 3′ TFLC5as:5′ CCCTGGTTTCTGCAGATACCATGCTAA (SEQ ID NO: 56) CC 3′ TFHC2s:5′ TACGACTCACTATAGGGCGAATTGG 3′ (SEQ ID NO: 57) TFLC2as1:5′ CCACAGATGCAGACAGGGAGGCAGGAGAC (SEQ ID NO: 58) TG 3′ TFLC1asR:5′ TTCGAAAAGTGTACTTACGTTTGATCTCCAG (SEQ ID NO: 59) CTTGGTACCAGCACCGAACG3′ TFLC2s: 5′ CCTGTCTGCATCTGTGGGAGATAGGGTCACC (SEQ ID NO: 60) ATCACATGC3′ TFLC4as: 5′ GATCTCCAGCTTGGTACCCTGACCGAACGTG (SEQ ID NO: 61) AATGG 3′TFLC3as: 5′ GTAGGCTGCTGATCGTGAAAGAAAAGTCTGT (SEQ ID NO: 62) GCCAGATCC 3′TFLC3s2: 5′ CACGATCAGCAGCCTACAGCCTGAAGATTTT (SEQ ID NO: 63)GTAAATTATTACTGTC 3′ TFLC08sds: 5′ GCAGCCTACAGCCTGAAGATTTTGCAACTTA (SEQID NO: 64) TTACTGTCAACAAG 3′ TFLC08sdsa:5′ CTTGTTGACAGTAATAAGTTGCAAAATCTTC (SEQ ID NO: 65) AGGCTGTAGGCTGC 3′LC105: 5′ CAGCAGCCTACAGCCTGAAGATTTTGCAAAT (SEQ ID NO: 66) TATTACTGTCAAC3′ LC103: 5′ GTTGACAGTAATAATTTGCAAAATCTTCAGG (SEQ ID NO: 67)CTGTAGGCTGCTG 3′ LC115: 5′ CAGTGGATCTGGCACAAAGTTTTCTTTCACG (SEQ ID NO:68) ATCAGCAGC 3′ LC113: 5′ GCTGCTGATCGTGAAAGAAAACTTTGTGCCA (SEQ ID NO:69) GATCCACTG 3′ LC125a: 5′ CTGCAGAAACCAGGGCAATCTCCTCAGCTCC (SEQ ID NO:70) TG 3′ LC123a: 5′ CAGGAGCTGAGGAGATTGCCCTGGTTTCTGC (SEQ ID NO: 71) AG3′

FIG. 14 shows hOAT (humanized cH36-IgG1) constant region sequences ofthe light (FIG. 14A) (SEQ ID NO: 97) and heavy chain (FIG. 14B) (SEQ IDNO: 98). FIG. 15 shows hFAT (humanized cH36-IgG4) constant regionsequences of the light (FIG. 15A) (SEQ ID NO: 99) and heavy chain (FIG.15B) (SEQ ID NO: 100). In each figure, the last amino acid residue ofthe framework 4 (FR4) variable region is connected to the first aminoacid residue of the constant region for hOAT and hFAT.

EXAMPLE 10 Expression and Purification of Humanized Anti-TF Antibodies

The partially humanized or fully humanized LC and HC clones were clonedinto expression vectors. The plasmid tKMC180 (see FIGS. 10A-B) was usedto express LC mutants fused to human kappa chain, and pJRS 355 (seeFIGS. 9A-B) or pLAM 356 (see FIGS. 9C-D) vector was used to express HCmutants fused to Fc of human IgG1 or IgG4. Some combinations of the HCand LC clones were then co-transfected into COS cells. The transientlyexpressed IgGs in COS cells were assayed for the whole IgG productionand binding to TF by ELISA.

The final fully-humanized forms of the anti-TF heavy and light variableregions (combination of HC08 and LC09) were cloned into Sunol's Megaexpression vector (pSUN34, see FIG. 11) and transfected into CHO and NSOcells for IgG expression. Stably transfected cell lines producing theIgG4κ or IgG1κ humanized anti-TF antibody were cloned. The selectedstable cell lines were then used to produce amounts of humanized anti-TFsufficient for analysis. The resulting humanized versions areapproximately 95% human in origin (the CDR sequences are notconsidered). The humanized IgG4 kappa version is designated hFAT(humanized IgG Four Anti-Tissue Factor antibody) and the IgG1 kappaversion is designated hOAT (humanized IgG One Anti-Tissue Factorantibody). These fully humanized versions of cH36 are intended fortreating chronic indications, such as cancer and inflammatory diseases.

One of the NSO cell lines (OAT-NSO-P10A7) that expresses hOAT(combination of HC08 and LC09) was thawed and extended in 10 mL of IMDMmedium supplemented with 10% FBS in a 15 mL tube and centrifuged. Thecell pellet was resuspended in 10 mL of fresh media and passed to a T25flask and incubated at 37° C. in 5% CO₂. In order to prepare asufficient number of cells to inoculate a hollow fiber bioreactor, thecells were expanded to obtain a total of 6×10⁸ cells. A bioreactor wasset up as per manufacturer's instruction manual. The harvested cellswere pelleted and resuspended in 60 mL of IMDM containing 35% FBS andinjected into the extracapillary space of the bioreactor. Concentrationsof glucose and lactate were monitored daily and the harvest material wascentrifuged and pooled. The harvested material was tested for anti-TFantibody concentrations by ELISA assay. The pooled sample containinganti-TF antibody (hOAT) were then purified and analyzed as describedbelow.

A. rProtein A Sepharose Fast Flow Chromatography

Recombinant humanized anti-TF monoclonal antibody consists of two lightand two heavy chains. Heavy chain is a fusion of mouse variable region(unaltered or humanized as described above) and human IgG1 or IgG4 Fcdomain, while light chain contains mouse variable region (unaltered orhumanized as described above) and human K domain. It is well establishedthat human IgG Fc region has high affinity for Protein A or recombinantProtein A (rProtein A).

Harvest pools containing humanized anti-TF antibody (hOAT) were adjustedto pH 8.0±0.1 by adding 0.08 ml of 1 M Tris-HCl, pH 8.0 per ml ofsample. Then the sample is filtered through low protein-binding 0.22micron filters (e.g., Nalgene sterile disposable tissue culture filterunits with polyethersulfone membrane from Nalge Nunc International, Cat.No. 167-0020). Following sample application, rProtein A column (fromPharmacia) is washed with 5 bed volumes of 20 mM Tris-HCl, pH 8.0 toremove unbound materials such as media proteins. Since the harvestmedium contains high content of bovine serum, a stepwise pH gradientwash was used to remove bovine IgG from the column. The stepwise pHgradient was achieved by increasing the relative percentage of Buffer B(100 mM acetic acid) in Buffer A (100 mM sodium acetate). A typical pHstepwise wash employed 20%, 40%, and 60% Buffer B. Elute the column with100% Buffer B and collect fractions b ased on A₂₈₀. The pooled fractionswere adjusted to pH 8.5 with addition of 1 M Tris base.

B. Q Sepharose Fast Flow Chromatography

Anion ion exchange chromatography is very effective in separatingproteins according to their charges. The eluted and pH-adjusted samplefrom rProtein A column was diluted with two volumes of water, and the pHis checked and adjusted to 8.5. The sample was then loaded to a 5 ml(1.6×2.5 cm) Q Sepharose Fast Flow equilibrated with 20 mM Tris-HCl, pH8.5 and the column washed with (1) 5 bed volumes of 20 mM Tris-HCl, pH8.5; and (2) 4 bed volumes of 20 mM Tris-HCl, p H 8.5 containing 100 m MNaCl. The IgG protein was then eluted with bed volumes of 20 mMTris-HCl, pH 8.5 containing 500 mM NaCl. The protein peaks were pooledand buffer-exchanged into PBS using ultrafiltration device.

Using the same transfection, cell culture, and purification methods,hFAT was also produced and purified.

EXAMPLE 11 Properties of Humanized Anti-TF Antibodies

A. Inhibition of TF Function by Humanized Anti-TF Antibody

One of the key properties of anti-TF antibodies is its ability toinhibit tissue factor-initiated blood coagulation. The purified hOAT andhFAT were measured for their ability to inhibit TF activity in astandard PT assay. PT assay is widely used to measure tissuefactor-dependent blood clotting times. The principal of this assay isthat tissue factor (TF) forms complex with factor VIIa in plasma. Thiscomplex then activates factor X to FXa; FXa then converts prothrombin tothrombin in the presence of factor Va and phospholipids. Thrombineventually leads to formation of a blood clot. In standard PT assays,lipidated TF is added to plasma to initiate blood coagulation and theclotting is recorded by an Organon Teknika Coag-A-Mate CoagulationAnalyzer or equivalent.

The anti-TF antibody, H36, inhibits human TF activity by a uniquemechanism. It binds to TF (free or in complex with factor VIIa) in sucha way that factor X and IX binding to TF:VIIa complex is prohibited,thus FX and FIX activation by TF:VIIa is blocked (see U.S. Pat. No.5,986,065). In PT tests, the prolongation of clotting times anti-TFantibody added into human plasma is a clear indication that thisTF-dependent coagulation is inhibited. The clotting time is related tothe amount of TF activity. A TF standard curve is generated by measuringPT clotting times of serially diluted TF. From the data of TF standardcurve, the inhibition of TF activity by anti-TF antibody is determined.

Reagents: Innovin (Cat No 68100-392) and Ci-Trol Coagulation Control,Level I (Cat No 68100-336) are obtained from VWR. Lipidated recombinanthuman TF was produced as described in Example 3.

Method: PT test is performed at 37 C using a Coagulation Analyzer. PTreaction is initiated by adding 0.2 ml of lipidated recombinant humantissue factor (e.g., Innovin) into 0.1 ml of human plasma (Ci-TrolControl Level I) containing 0.01 ml buffer (50 mM Tris-HCl, pH 7.5, 0.1%BSA) or anti-TF antibody.

-   -   1. Add purified water to a vial of Innovin according to        manufacturer's instruction. Warm the reagent to 37° C. The        reagent is stable for a few days if stored at 4-8° C.    -   2. Add I ml purified water to each vial of Ci-Trol. Mix to        solubilize. If more one vials are used, combine them into one        container (e.g., a 10 ml test tube). 1 ml Ci-Trol can run 5        assays (each assay uses 2×0.1 ml=0.2 ml). Ci-Trol can be stored        on ice and last for a few hours.    -   3. From anti-TF antibody stock, make a series of anti-TF        antibody solutions (200 nM to 1600 nM) with 50 mM Tris-HCl, pH        7.5, 0.1% BSA    -   4. Add 10 μl of 50 mM Tris-HCl, pH 7.5, 0.1% BSA or 10 μl of        diluted anti-TF to each well of the twin-well cuvette that        contains 0.1 ml of Ci-Trol. Use a pipette with 0.1 ml tip to mix        each well. Make sure no air bubbles are in the well. Following        mixing anti-TF (or buffer) with plasma (Ci-Trol), measure        clotting times within 10 min by adding 0.2 ml of Innovin to the        plasma.

5. For TF standard curve, first dilute Innovin (100% TF) to 20%, 10%,5%and 2.5% with 50 mM Tris-HCl, pH 7.5, 0.1% BSA. Then PT assays wereperformed as in Step 4 but using diluted Innovin samples.

Table 3 is the summary of the effect of cH36, hOAT, and hFAT on PTclotting times. Compared to the data in Table 4, cH36, hFAT, and hOATshowed very potent inhibition of TF function. At a protein concentrationof above 12.9 nM, all antibodies achieved about 95% inhibition. Theresults in Table 3 also indicate that humanization of anti-TF, cH36, bythe method described above did not have any significant effect on cH36inhibitory activity since both hFAT and hOAT showed very similar abilityto inhibit TF-dependent blood coagulation as seen for cH36. TABLE 3Effect on Prothrombin Times by Chimeric (cH36) and Humanized) Anti-TFAntibodies (hFAT and hOAT)^(#) Anti-TF Antibody Concentrations (nM) inPT Time (in seconds) PT Assays cH36 hOAT hFAT 0 12.2 12.2 12.2 6.45 14.9nd nd 9.7 17.8 16.5 nd 12.9 19.8 18.9 20.5 25.8 40 33.7 41.7 51.6 101.382.1 94.8^(#)All assays used the same 100% TF activity (concentration) sample asin Table 4.

TABLE 4 Clotting Times and Relative Tissue Factor Activities(Concentrations) Relative TF Activities (Concentrations) PT ClottingTimes (Seconds) 100% (neat) 11.90  20% 13.225  10% 14.675  5% 16.700 2.5% 20.000

B. Determination of Affinity Constants

The affinity of humanized anti-TF antibody for TF was determined bysurface plasmon resonance (BIAcore from Pharmacia Biosensor) withrecombinant human tissue factor covalently immobilized on a CM5 sensorchip. The affinity constants were the average data calculated from fouranti-TF monoclonal antibody concentrations (0.125 nM, 0.25 nM, 0.5 nM,and 1 nM) by the BIAcore computer software. The results in Table 5indicate that humanization of anti-TF, cH36, by the method describedabove did not have any significant effect on cH36 affinity for TF sinceboth cH36 and hFAT have similar affinity for TF. TABLE 5 ApparentAffinity and Dissociation Constants of Anti-TF Antibodies Anti-TFAntibody Apparent K_(a) (M⁻¹) Apparent K_(d) (M) H36 1.56 × 10¹⁰  6.4 ×10⁻¹¹ cH36 7.94 × 10⁹  1.26 × 10⁻¹⁰ hFAT 2.99 × 10⁹  3.35 × 10⁻¹⁰

The invention has been described in detail with reference to preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of the disclosure, may make modificationand improvements within the spirit and scope of the invention.

1. A humanized antibody that binds specifically to human tissue factor(TF) to form a complex, wherein factor X or factor IX binding to thecomplex and the FX or FIX activation by TF:VIIa are inhibited.
 2. Thehumanized antibody of claim 1, wherein the antibody has a dissociationconstant (K_(d)) for the TF of less than about 0.5 nM.
 3. The humanizedantibody of claim 1 or 2, wherein the antibody is further characterizedby increasing blood clotting time by at least about 5 seconds asdetermined by a standard prothrombin (PT) clotting assay at an antibodyconcentration of <15 nM.
 4. The humanized antibody of claim 1 or 2,wherein the antibody has a binding specificity for the TF about equal orgreater than the antibody obtained from cell line H36.D2.B7 depositedunder ATCC Accession No. HB-12255.
 5. The humanized antibody of claim 1or 2, wherein the antibody has a binding affinity for the TF about equalto or greater than the antibody obtained from cell line H36.D2.B7deposited under ATCC Accession No. HB-12255.
 6. The humanized antibodyof claim 1 or 2, wherein the antibody comprises at least one fullymurine complimentarity determining region (CDR).
 7. The humanizedantibody of claim 1 or 6, wherein the antibody comprises at least onefully human framework (FR) region.
 8. The humanized antibody of claim 1,wherein the antibody has at least about 90% amino acid sequence identityto a human antibody.
 9. The humanized antibody of claim 1, wherein thevariable region of the humanized antibody has at least about 70% aminoacid sequence identity to a human antibody variable region.
 10. Thehumanized antibody of claim 1, wherein each of frameworks (FRs) 1, 2, 3and 4 has at least about 95% amino acid sequence identity to the lightchain FR sequences shown in FIG. 12A (SEQ ID NO: 79).
 11. The humanizedantibody of claim 1, wherein the antibody comprises a light chainconstant region having at least about 95% amino acid sequence identityto the sequence shown in FIG. 14A or 15A (SEQ ID NOS: 97 or 99).
 12. Thehumanized antibody of claim 1, wherein each of frameworks (FRs) 1, 2, 3and 4 has at least about 95% amino acid sequence identity to the heavychain sequences shown in FIG. 13A (SEQ ID NO: 91).
 13. The humanizedantibody of claim 12, wherein the antibody further comprises a heavychain constant region having at least about 95% amino acid sequenceidentity to sequence shown in FIG. 14B or 15B (SEQ ID NOS: 98 or 100).14. The humanized antibody of claim 1, wherein the antibody has an IgG1(hOAT) or IgG4 (hFAT) isotype.
 15. A human TF binding fragment of thehumanized antibody of claim
 1. 16. The human TF binding fragment ofclaim 15, wherein the fragment is Fab, Fab′, or F(ab)₂.
 17. A humanizedantibody comprising at least one murine complementarity determiningregion (CDR), wherein the antibody binds specifically to human tissuefactor (TF) to form a complex, and further wherein factor X or factor IXbinding to TF or TF:FVIIa and activation by TF:FVIIa thereto isinhibited.
 18. The humanized antibody of claim 17, wherein all the CDR(light and heavy chain) are murine.
 19. The humanized antibody of claim17, wherein the antibody further comprises as least one human framework(FR) region.
 20. The humanized antibody of claim 19, wherein the aminoacid sequences of all the FR (light and heavy chain) are human or within2 amino acid substitutions of being human.
 21. The humanized antibody ofclaim 17, wherein the first CDR (CDR1) of the heavy chain hypervariableregion is at least 95% identical to the CDR1 amino acid sequence shownin FIG. 13B (SEQ ID NO: 8).
 22. The humanized antibody of claim 17,wherein the second CDR (CDR2) of the heavy chain hypervariable region isat least 95% identical to the CDR2 amino acid sequence shown in FIG. 13C(SEQ ID NOS: 9 or 101).
 23. The humanized antibody of claim 17, whereinthe third CDR (CDR3) of the heavy chain hypervariable region is at least95% identical to the CDR3 amino acid sequence shown in FIG. 13D (SEQ IDNO: 10).
 24. The humanized antibody of claim 17, wherein the first CDR(CDR1) of the light chain hypervariable region is at least 95% identicalto the CDRI amino acid sequence shown in FIG. 12B (SEQ ID NO: 2). 25.The humanized antibody of claim 17, wherein the second CDR (CDR2) of thelight chain hypervariable region is at least 95% identical to the CDR2amino acid sequence shown in FIG. 12C (SEQ ID NO: 6).
 26. The humanizedantibody of claim 17, wherein the third CDR (CDR3) of the light chainhypervariable region is at least 95% identical to the CDR3 amino acidsequence shown in FIG. 12D (SEQ ID NO: 7).
 27. The humanized antibody ofclaim 19, wherein the first framework (FR1) of the heavy chainhypervariable region is at least 95% identical to the FR1 amino acidsequence shown in FIG. 13A (SEQ ID NO: 91).
 28. The humanized antibodyof claim 27, wherein the FR1 comprises at least one of the followingamino acid changes: El to Q; Q5 to V; P9 to G; L11 to V; V12 to K; Q19to R; and T24 to A.
 29. The humanized antibody of claim 19, wherein thesecond framework (FR2) of the heavy chain hypervariable region is atleast 95% identical to the FR2 amino acid sequence shown in FIG. 13A(SEQ ID NO: 91).
 30. The humanized antibody of claim 29, wherein the FR2comprises at least one of the following amino acid changes: 41H to P;and 44S to G.
 31. The humanized antibody of claim 19, wherein the thirdframework (FR3) of the heavy chain hypervariable region is at least 95%identical to the FR3 amino acid sequence shown in FIG. 13A (SEQ ID NO:91).
 32. The humanized antibody of claim 31, wherein the FR3 comprisesat least one of the following amino acid changes: 76S to T; 77T to S;80F to Y; 82H to E; 84N to S; 87T to R; 89D to E; and 91S to T.
 33. Thehumanized antibody of claim 19, wherein the fourth framework (FR4) ofthe heavy chain hypervariable region is at least 95% identical to theFR4 amino acid sequence shown in FIG. 13A (SEQ ID NO: 91).
 34. Thehumanized antibody of claim 33, wherein the FR4 comprises the followingamino acid change: 113L to V.
 35. The humanized antibody of claim 19,wherein the first framework (FR1) of the light chain hypervariableregion is at least about 95% identical to the FR1 amino acid sequenceshown in FIG. 12A (SEQ ID NO: 79).
 36. The humanized antibody of claim35, wherein the FR1 comprises at least one of the following amino acidchanges: 11QL to L; 15L to V; 17E to D; and 18 to R.
 37. The humanizedantibody of claim 19, wherein the second framework (FR2) of the lightchain hypervariable region is at least about 95% identical to the FR2amino acid sequence shown in FIG. 12A (SEQ ID NO: 79).
 38. The humanizedantibody of claim 37, wherein the FR2 has the following amino acidchanges: 37Q to L.
 39. The humanized antibody of claim 19, wherein thethird framework (FR3) of the light chain hypervariable region is atleast about 95% identical to the FR3 amino acid sequence shown in FIG.12A (SEQ ID NO: 79).
 40. The humanized antibody of claim 39, wherein theFR3 has the following amino acid changes: 70K to D, 74K to T, 80A to P,84A to V, and 85N to T.
 41. The humanized antibody of claim 40, whereinthe fourth framework (FR4) of the light chain hypervariable region is atleast about 95% identical to the FR4 amino acid sequence shown in FIG.12A (SEQ ID NO: 79).
 42. The humanized antibody of claim 41, wherein theFR4 comprises the following amino acid changes: 100A to Q; and 106L toI.
 43. A human TF binding fragment of the humanized antibody of claim17.
 44. The human TF binding fragment of claim 43, wherein the fragmentis Fab, Fab′, or F(ab)₂.
 45. A humanized antibody comprising at leastone murine complementarity determining region (CDR), wherein theantibody binds specifically to human tissue factor (TF) to form acomplex, and further wherein factor X or factor IX binding to TF orTF:FVIIa and activation by TF:FVIIa thereto is inhibited, the antibodycomprising on the heavy chain: a) a first CDR (CDR1) which is at least95% identical to CDR1 amino acid sequence shown in FIG. 13B (SEQ ID NO:8), b) a second CDR (CDR2) which is at least 95% identical to the CDR2amino acid sequence shown in FIG. 13C (SEQ ID NOS: 9 or 101), c) a thirdCDR (CDR3) which is at least 95% identical to the CDR3 amino acidsequence shown in FIG. 13D (SEQ ID NO: 10), d) a first framework (FR1)which is at least 95% identical to the FR1 amino acid sequence shown inFIG. 12A (SEQ ID NO: 79), e) a second framework (FR2) which is at least95% identical to the FR2 amino acid sequence shown in FIG. 12A (SEQ IDNO: 79), f) a third framework (FR3) which is at least 95% identical tothe FR3 amino acid sequence shown in FIG. 12A (SEQ ID NO: 79), and g) afourth framework (FR4) which is at least 95% identical to the FR4 aminoacid sequence shown in FIG. 12A (SEQ ID NO: 79).
 46. The antibody ofclaim 45 further comprising on the light chain, h) a first CDR (CDR1)which is at least 95% identical to CDR1 amino acid sequence shown inFIG. 12B (SEQ ID NO: 2), i) a second CDR (CDR2) which is at least 95%identical to the CDR2 amino acid sequence shown in FIG. 12C (SEQ ID NO:6), j) a third CDR (CDR3) which is at least 95% identical to the CDR3amino acid sequence shown in FIG. 12C (SEQ ID NO: 6), k) a firstframework (FR1) which is at least 95% identical to the FR1 amino acidsequence shown in FIG. 12A (SEQ ID NO: 79), l) a second framework (FR2)which is at least 95% identical to the FR2 amino acid sequence shown inFIG. 12A (SEQ ID NO: 79), m) a third framework (FR3) which is at least95% identical to the FR3 amino acid sequence shown in FIG. 12A (SEQ IDNO: 79), and n) a fourth framework (FR4) which is at least 95% identicalto the FR4 amino acid sequence shown in FIG. 12A (SEQ ID NO: 79). 47.The antibody of claim 45 further comprising the light chain constantsequence of FIG. 14A (SEQ ID NO: 97) or FIG. 15A (SEQ ID NO: 99). 48.The antibody of claim 45 further comprising the heavy chain constantregion of FIG. 14B (SEQ ID NO: 98) or FIG. 15B (SEQ ID NO: 100).
 49. Ahuman TF binding fragment of the humanized antibody of claim
 45. 50. Thehuman TF binding fragment of claim 45, wherein the fragment is Fab,Fab′, or F(ab)₂.
 51. A humanized antibody comprising on the heavy chain:a) a first CDR (CDR1) identical to the CDR1 amino acid sequence shown inFIG. 13B (SEQ ID NO: 8), b) a second CDR (CDR2) identical to the CDR2amino acid sequence shown in FIG. 13C (SEQ ID NOS: 9 or 101), c) a thirdCDR (CDR3) identical to the CDR3 amino acid sequence shown in FIG. 13D(SEQ ID NO: 10), d) a first framework (FR1) identical to the FR1 aminoacid sequence shown in FIG. 13A (SEQ ID NO: 91), e) a second framework(FR2) identical to the FR2 amino acid sequence shown in FIG. 13A (SEQ IDNO: 91), f) a third framework (FR3) identical to the FR3 amino acidsequence shown in FIG. 13A (SEQ ID NO: 91); and g) a fourth framework(FR4) identical to the FR4 amino acid sequence shown in FIG. 13A (SEQ IDNO: 91); and on the light chain: h) a first CDR (CDR1) identical to CDR1amino acid sequence shown in FIG. 12B (SEQ ID NO: 2), i) a second CDR(CDR2) identical to the CDR2 amino acid sequence shown in FIG. 12C (SEQID NO: 6), j) a third CDR (CDR3) identical to the CDR3 amino acidsequence shown in FIG. 12D (SEQ ID NO: 7), k) a first framework (FR1)identical to the FR1 amino acid sequence shown in FIG. 12A (SEQ ID NO:79), l) a second framework (FR2) identical to the FR2 amino acidsequence shown in FIG. 12A (SEQ ID NO: 79), m) a third framework (FR3)identical to the FR3 amino acid sequence shown in FIG. 12A (SEQ ID NO:79), and n) a fourth framework (FR4) identical to the FR4 amino acidsequence shown in FIG. 12A (SEQ ID NO: 79).
 52. The antibody of claim 51further comprising the light chain constant sequence of FIG. 14A (SEQ IDNO: 97) or FIG. 15A (SEQ ID NO: 99).
 53. The antibody of claim 51further comprising the heavy chain constant sequence of FIG. 14B (SEQ IDNO: 98) or 15B (SEQ ID NO: 100).
 54. The humanized antibody of claim 51,wherein the antibody has an IgG1 or IgG4 isotype.
 55. A human TF bindingfragment of the humanized antibody of claim
 4. 56. The human TF bindingfragment of claim 55, wherein the fragment is Fab, Fab′, or F(ab)₂. 57.The humanized antibody of claim 1, wherein the antibody is a monoclonalantibody.
 58. A single-chain antibody comprising the hypervariableregion of the antibody of claim
 1. 59. An isolated nucleic acid encodingat least one of the heavy or light chain of the humanized antibody ofclaim
 1. 60. A recombinant vector comprising the isolated nucleic acidof claim
 59. 61. A host cell comprising the recombinant vector of claim60.
 62. A composition comprising the humanized antibody of claim 1, andat least one pharmaceutically acceptable carrier.
 63. A method ofinhibiting blood coagulation in a mammal, the method comprisingadministering to the mammal an effective amount of the humanizedantibody of claim 1 or fragment thereof that binds specifically to humantissue factor (TF) to form a complex, wherein factor X or factor IXbinding to TF or TF:FVIIa and activation by TF:FVIIa thereto isinhibited, the method further comprising forming a specific complexbetween the antibody and the TF to inhibit the blood coagulation.
 64. Amethod of inhibiting blood coagulation in a mammal, the methodcomprising administering to the mammal, an effective amount of thehumanized antibody of claim 7 comprising at least or fragment thereof,wherein the antibody or fragment binds specifically to human tissuefactor (TF) to form a complex, and further wherein factor X or factor IXbinding to TF or TF:FVIIa and activation by TF:FVIIa thereto isinhibited, the method further comprising forming a specific complexbetween the antibody and the TF to inhibit the blood coagulation.
 65. Amethod of inhibiting blood coagulation in a mammal, the methodcomprising administering to the mammal, an effective amount of ahumanized antibody or fragment thereof wherein the antibody bindsspecifically to human tissue factor (TF) to form a complex, and furtherwherein factor X or factor IX binding to TF or TF:FVIIa and activationby TF:FVIIa thereto is inhibited, the antibody or fragment comprising onthe heavy chain: a) a first CDR (CDR1) which is at least 95% identicalto CDR1 amino acid sequence shown in FIG. 13B (SEQ ID NO: 8), b) asecond CDR (CDR2) which is at least 95% identical to the CDR2 amino acidsequence shown in FIG. 13C (SEQ ID NOS: 9 or 101), c) a third CDR (CDR3)which is at least 95% identical to the CDR3 amino acid sequence shown inFIG. 13D (SEQ ID NO: 10), d) a first framework (FR1) which is at least95% identical to the FR1 amino acid sequence shown in FIG. 13A (SEQ IDNO: 91), e) a second framework (FR2) which is at least 95% identical tothe FR2 amino acid sequence shown in FIG. 13A (SEQ ID NO: 91), f) athird framework (FR3) which is at least 95% identical to the FR3 aminoacid sequence shown in FIG. 13A (SEQ ID NO: 91), g) a fourth framework(FR4) which is at least 95% identical to the FR4 amino acid sequenceshown in FIG. 13A (SEQ ID NO: 91); and on the light chain, h) a firstCDR (CDR1) which is at least 95% identical to CDR1 amino acid sequenceshown in FIG. 12B (SEQ ID NO: 2), i) a second CDR (CDR2) which is atleast 95% identical to the CDR2 amino acid sequence shown in FIG. 12C(SEQ ID NO: 6), j) a third CDR (CDR3) which is at least 95% identical tothe CDR3 amino acid sequence shown in FIG. 12D (SEQ ID NO: 7), k) afirst framework (FR1) which is at least 95% identical to the FR1 aminoacid sequence shown in FIG. 12A (SEQ ID NO: 79, l) a second framework(FR2) which is at least 95% identical to the FR2 amino acid sequenceshown in FIG. 12A (SEQ ID NO: 79), m) a third framework (FR3) which isat least 95% identical to the FR3 amino acid sequence shown in FIG. 12A(SEQ ID NO: 79), n) a fourth framework (FR4) which is at least 95%identical to the FR4 amino acid sequence shown in FIG. 12A (SEQ ID NO:79), o) a light chain constant region which is at least 95% identical tothe amino acid sequence shown in FIG. 14A (SEQ ID NO: 97) or FIG. 15A(SEQ ID NO: 99), and p) a heavy chain constant region which is at least95% identical to the amino acid sequence shown in FIG. 14B (SEQ ID NO:98) or FIG. 15B (SEQ ID NO: 100).
 66. A method of inhibiting bloodcoagulation in a mammal, the method comprising administering to themammal, an effective amount of a humanized antibody or fragment thereofwherein the antibody binds specifically to human tissue factor (TF) toform a complex, and further wherein factor X or factor IX binding to TFor TF:FVIIa and activation by TF:FVIIa thereto is inhibited, theantibody or fragment comprising on the heavy chain: a) a first CDR(CDR1) identical to CDR1 amino acid sequence shown in FIG. 13B (SEQ IDNO: 8), b) a second CDR (CDR2) identical to the CDR2 amino acid sequenceshown in FIG. 13C (SEQ ID NOS: 9 or 101), c) a third CDR (CDR3)identical to the CDR3 amino acid sequence shown in FIG. 13D (SEQ ID NO:10), d) a first framework (FR1) identical to the FR1 amino acid sequenceshown in FIG. 13A (SEQ ID NO: 91), e) a second framework (FR2) identicalto the FR2 amino acid sequence shown in FIG. 13A (SEQ ID NO: 91), f) athird framework (FR3) identical to the FR3 amino acid sequence shown inFIG. 13A (SEQ ID NO: 91), g) a fourth framework (FR4) identical to theFR4 amino acid sequence shown in FIG. 13A (SEQ ID NO: 91); and on thelight chain: h) a first CDR (CDR1) identical to CDR1 amino acid sequenceshown in FIG. 12B (SEQ ID NO: 2), i) a second CDR (CDR2) identical tothe CDR2 amino acid sequence shown in FIG. 12C (SEQ ID NO: 6), j) athird CDR (CDR3) identical to the CDR3 amino acid sequence shown in FIG.12D (SEQ ID NO: 7), k) a first framework (FR1) identical to the FR1amino acid sequence shown in FIG. 12A (SEQ ID NO: 79), l) a secondframework (FR2) identical to the FR2 amino acid sequence shown in FIG.12A (SEQ ID NO: 79), m) a third framework (FR3) identical to the FR3amino acid sequence shown in FIG. 12A (SEQ ID NO: 79), n) a fourthframework (FR4) identical to the FR4 amino acid sequence shown in FIG.12A (SEQ ID NO: 79), o) a light chain constant region which is identicalto the amino acid sequence shown in FIG. 14A (SEQ ID NO: 97) or FIG. 15A(SEQ ID NO: 99), and p) a heavy chain constant region which is identicalto the amino acid sequence shown in FIG. 14B (SEQ ID NO: 98) or FIG. 15B(SEQ ID NO: 100).
 67. A method of detecting tissue factor (TF) in abiological sample, the method comprising contacting a biological samplewith the antibody of claim 1 under conditions conducive to forming acomplex and detecting the complex as being indicative of the TF in thebiological sample.
 68. A method for producing the humanized antibody ofclaim 1, wherein the method comprises providing a host cell transformedwith either 1) a first expression vector encoding the light chain of thehumanized antibody or fragment thereof and a second expression vectorencoding the heavy chain of the humanized antibody or fragment thereof,or 2) a single expression vector encoding both the light chain and theheavy chain of the humanized antibody or fragment thereof, maintainingthe host cell under growth conditions in which each chain is expressed;and isolating the humanized antibody or fragment thereof.
 69. A methodfor producing a humanized antibody, wherein the method comprises: a)comparing the amino acid sequence of a light chain framework from arodent antibody against a collection of corresponding human antibodyframework sequences, b) selecting a human framework sequence from thecollection having the greatest amino acid sequence identity to thecorresponding rodent light chain framework, c) mutagenizing a DNAsegment encoding the rodent light chain framework to encode a humanizedlight chain framework having an amino acid sequence that issubstantially identical to the human framework sequence selected in stepb), d) repeating steps a) thru c) for each individual framework of therodent light chain to produce a plurality of DNA sequences in which eachsequence encodes a humanized light chain framework, wherein each of thecorresponding human framework sequences selected in step b) are from thesame or different human antibody, e) assembling into a first vectorencoding at least the light chain variable region of the rodentantibody, the DNA sequences encoding the humanized framework sequencesproduced in step d); and f) introducing the assembled vector into a hostunder conditions sufficient to produce the humanized antibody.
 70. Themethod of claim 69, wherein the method further comprises: g) comparingthe amino acid sequence of a heavy chain framework from the rodentantibody against a collection of corresponding human antibody frameworksequences, h) selecting a human framework sequence from the collectionhaving the greatest amino acid sequence identity to the correspondingrodent heavy chain framework, i) mutagenizing a DNA segment encoding therodent heavy chain framework to encode a humanized heavy chain frameworkhaving an amino acid sequence that is substantially identical to thehuman framework sequence selected in step h); and j) repeating steps g)thru i) for each individual framework of the rodent heavy chain toproduce a plurality of DNA sequences in which each sequence encodes ahumanized heavy chain framework, wherein each of the corresponding humanframework sequences selected in step h) are from the same or differenthuman antibody.
 71. The method of claim 70, wherein the method furthercomprises assembling into a second vector encoding at least the heavychain variable region of the rodent antibody, the DNA sequences encodingthe humanized framework sequences produced in step j); and introducingthe assembled first and second vectors into a host under conditionssufficient to produce the humanized antibody.
 72. The method of claim70, wherein the method further comprises assembling into the firstvector encoding at least the light chain variable region of the rodentantibody, the DNA sequences encoding the humanized framework sequencesproduced in step j); and introducing the assembled first vector into ahost under conditions sufficient to produce the humanized antibody.